Recombinant TPO Research Articles

Recombinant human thrombopoietin (rhTPO) of different dosing regimens for refractory/relapsed primary immune thrombocytopenia: a multicenter, randomized controlled trial and pharmacokinetics study

Recombinant human TPO (rhTPO) is effective for refractory/relapsed primary immune thrombocytopenia (ITP), but optimal dosing regimen remains elusive. In this multicenter, randomized, controlled trial, a total of 282 adult ITP patients (mean age 47.3 years; 82 men) with a platelet count ≤30 × 109/L or >30 × 109/L with active bleeding randomly received a once daily (QD) subcutaneous injection of 7500 U (n = 64) or 15000 U rhTPO for 14 injections, or 15000 U or 30000 U rhTPO once every other day (QOD) for 7 injections. The primary outcomes included change from baseline in platelet count and total response rate (TRR) on day 14. On day 14, the median increase of platelet count from baseline was the highest in the 15000-U QD group (167.5 × 109/L, interquartile range [IQR] 23.0–295.0 × 109/L), followed by the 30000-U QOD group (57.5 × 109/L, IQR 9.0–190.0 × 109/L) (ANCOVA P < .001; P = .266 with baseline count as a covariate). The TRR on day 14 was also the highest in the 15000-U QD group (63.2%), followed by the 30000-U QOD group (59.7%). The rate of grade 3 and above adverse events did not differ among the four groups. There were no new safety concerns. All 4 regimens are safe and well-tolerated. The 30000-U QOD regimen is practically indistinguishable in efficacy to the 15000-U QD regimen.

Eltrombopag Causes Cell Cycle Arrest of T Cells through Iron Chelation and Could Modulate the Immune Response in ITP

Background Thrombopoietin receptor agonists (TPO-RAs) are the recommended second line treatment in most guidelines for immune thrombocytopenia (ITP) allowing patients to avoid immunosuppressive treatments (Provan et al and Neunert et al 2019). Three different TPO-RAs are licensed for use in ITP. Real world evidence has shown that if patients do not respond to one agent, they may respond to another. This is thought to relate to different signalling pathways, and possibly one TPO-RA creating a stronger signal than another (Olnes et al, 2012; D'Arena et al, 2013). Nonetheless, it is difficult to fully explain responses on switching agents, especially a response to Eltrombopag (ELT) when Romiplostim (ROMI) has failed. In addition to being a TPO-RA, ELT is a small molecule peptide, which chelates cations, in particular iron and calcium. We have previously shown its ability to shuttle iron out of cells, and the impact this has on liver, cardiac and megakaryocyte cells in vitro (Roth et al, 2012; Vlachodimitropoulou et al, 2016, Liu et al 2022). We previously reported that ELT has an anti-proliferative effect on stimulated Jurkat and primary T cells, and demonstrated that patients on ELT have a reduced frequency of terminally differentiated effector (TEMRA) CD8 T cells compared to patients on ROMI (Sayed et al, ASH, 2019). We propose that this is related to an intracellular iron chelation effect, which could either explain or be additive to its megakaryocyte stimulating effect in ITP. In this study, we further explored the immunomodulatory effect of ELT on CD8 T cells in ITP. Methods Primary T cells were stimulated with Dynabeads Human T-Activator CD3/CD28 in the presence of ELT, ROMI, Deferoxamine (DFX) or human recombinant TPO (rhTPO) at increasing doses based on their calculated therapeutic serum levels. DFX and rhTPO act as positive control for iron chelation and TPO-R, respectively. Cell cycle was measured using Propidium Iodide (PI) and analysed using flow cytometry. To explore the in vivo effect of ELT, we measured CD8 TEMRA cells before and after treatment in a cohort of patients with ITP treated with ELT. Results Primary T cells treated with ELT resulted in cell cycle arrest to G1 phase. This effect was more pronounced with increasing doses of ELT. DFX exhibited similar effect on primary T cells, however, the induction of G1 phase cell cycle arrest was less potent than ELT. ROMI and rhTPO did not affect cell cycle progression of primary T cells (Figure 1). To confirm whether this cell cycle arrest was due to the iron chelating properties of ELT, we treated the arrested primary T cells with ammonium iron (III) for 24 hours, and found that cell cycle was able to be rescued. We further investigated the immunomodulatory effect of ELT in patients with ITP by assessing CD8 TEMRA cells (CD45RA+CD62L-) before and after ELT treatment. We found that patients who responded to ELT had a reduction in the frequency of TEMRA cells correlating to their platelet count. An example shown in Figure 2 of a patient who had previously failed ROMI, Mycophenolate Mofetil (MMF) and Rituximab, and subsequently responded to ELT. Prior to ELT, at low platelet count, patient presented with high frequency of CD8 TEMRA cells. Response to ELT resulted in an increased platelet count and a corresponding fall in CD8 TEMRA cells. Discussion Here we show that ELT suppresses the proliferation of T cells, through cell cycle arrest via its iron chelating properties. We propose that this could have a dual impact in patients with ITP, increasing platelet production as well as dampening down the immune response, hence explaining why switching to ELT could be effective if other TPO-RAs have failed. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

IFN-γ Inhibitors and Ruxolitinib Therapy for Acquired Severe Aplastic Anemia in an Ex-Vivo Model

Background:Severe aplastic anemia (SAA) is a life-threatening disorder that is associated with multiple etiologies, both inherited and acquired. In acquired SAA, oligoclonal expansion of dysregulated CD8+ cytotoxic T cells, abnormal function of CD4+ T helper cells, along with elevated production of IFN-γ and TNF-α have been associated with the apoptosis of hematopoietic stem and progenitor cells (HSPC) (Young, N Engl J Med, 2018). Currently, the first line treatment for patients who have a suitable HLA matched donor is a hematopoietic progenitor cell transplant (HPCT). When HPCT is not possible, due to lack of a closely matched HLA donor and/or concomitant co-morbidities, then the treatment of choice is immunosuppression with anti-thymocyte globulin, cyclosporine and eltrombopag (ELT)(Georges et al, Blood, 2018). Alvarado et al (Blood, 2019) recently demonstrated that ELT bypasses the inhibitory effect of IFN-γ by alternatively activating TPO signaling. However, ELT cannot overcome other IFN-γ mediated effect through JAK-STAT1 phosphorylation or apoptosis via Fas/FasL. Alternative therapies are in great need for patients with aSAA as treatment response is sub-optimal.Objective:To determine the effects of IFN-γ neutralizing antibodies or Ruxolitinib on HSPCs survival, proliferation and differentiation in an ex vivo culture of human CD34+ cells in the presence of IFN-γ and TNF-α.Design/Methods:Human CD34+ HSPCs were isolated from cord blood, based on CD34 microbeads magnetic selection (Miltenyl Biotec, Germany). The CD34+ cells were cultured in StemSpan Serum-Free Medium II (STEMCELL Technologies) supplemented with 5 ng/mL human stem cell factor (SCF), FMS-like tyrosine kinase 3 ligand (Flt3L) and 5 ng/mL recombinant human TPO. A 1x10 5 CD34+ HSPCs were seeded in a 96 well plate. HSPC alone, with and without IFN-γ (100 ng/mL) and TNF-α (10 ng/mL), were the negative and positive control, respectively. Specific IFN-γ neutralizing antibody, B27 (BD Pharmingen), MD1 (BioLegend) and B133.5 (ImmunoTools) or Ruxolitinib (Jakafi, Incyte) were added to the culture and HSPC were harvested and assayed for their survival at day 7 and 14. Each experimental condition was set up in triplicate. The cells were cultured at 37°C with 5% CO 2. Also, we assessed the multi-lineage differentiation capacity with a selective colony forming units (CFU) assay. Fourteen days after co-culture of each experimental treatment, 500 CD34+ cells were seeded in 6-well plates Smart Dish™ (STEMCELL Technologies). Big burst forming units of erythroid (BFU-E), CFU of granulocyte and megakaryocyte (GM) and granulocyte, erythrocyte, macrophage, megakaryocyte (GEMM) were counted and compared between the experimental groups. The signaling pathways were determined using phospho flow-cytometric analysis of pSTAT1, pSTAT3, pSTAT5. All statistically analyzed data is represented as mean ± SD. Differences between groups were analyzed by multiple 2-tailed unpaired Student t tests using Excel. Statistically significant differences were represented as *P <0.05, ** P <0.01 and *** P<0.001.Results:The number of CD34+ cells at day 7 and day 14 of culture in the presence of IFN-γ and TNF-α was significantly lower (21% ± 3 (p<0.0006) and 15% ± 6 (p<0.02), respectively, than that of the control (Fig 1). Importantly, the myelosuppressive effect of IFN-γ and TNF-α was significantly rescued by the addition of Ruxolitinib (at day 14, p<0.05) or IFN-γ neutralizing antibodies (B27 and B133.5, respectively) (day 7 and 14, p<0.01). Improvement in HSPC survival ranged between 1.5-3.3 fold compared to our negative control. Based on the CFU analysis, the CD34+ cells cultured in the presence of B27, B133.5 or Ruxolitinib, were able to produce more CFU at day 7 (p<0.01, p<0.01 and p<0.05 respectively)(Fig 2A). Additionally, Both B27 (p < 0.01) and Ruxolitinb (p ≤ 0.001) were found to produce more CFU-GM on day 14 (Fig 2B). Phospho flow-cytometry demonstrated a significant decrease in STAT1 phosphorylation of CD34+ cells in the presence of B27 and B133.5 (p<0.05, p<0.001, respectively).Conclusions:Our preliminary studies supports the potential benefits of utilizing IFN-γ neutralizing antibodies or Ruxolitinib to improve HSPC survival, proliferation and differentiation in aSAA. Future studies will need to be done to investigate the exact mechanisms of action and the effects of IFN-γ neutralizing antibodies in an animal model of aSAA. [Display omitted] DisclosuresCairo: Amgen: Speakers Bureau; Jazz Pharmaceutical: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Sanofi: Speakers Bureau; Servier: Speakers Bureau; Sobi: Speakers Bureau. OffLabel Disclosure:Ruxolitinib was used to inhibit JAK-STAT signaling pathway in an Ex-Vivo model of Aplastic Anemia. Drug wasn't supplied by drug company.

Thrombocytopenia in Patients with Chronic Hepatitis C Virus Infection.

Thrombocytopenia in patients with chronic hepatitis C virus (HCV) infection is a major problem. The pathophysiology is multifactorial, with auto-immunogenicity, direct bone marrow suppression, hypersplenism, decreased production of thrombopoietin and therapeutic adverse effect all contributing to thrombocytopenia in different measures. The greatest challenge in the care of chronic HCV patients with thrombocytopenia is the difficulty in initiating or maintaining IFN containing anti-viral therapy. Although at present, it is possible to avoid this challenge with the use of the sole Direct Antiviral Agents (DAAs) as the primary treatment modality, thrombocytopenia remains of particular interest, especially in cases of advanced liver disease. The increased risk of bleeding with thrombocytopenia may also impede the initiation and maintenance of different invasive diagnostic and therapeutic procedures. While eradication of HCV infection itself is the most practical strategy for the remission of thrombocytopenia, various pharmacological and non-pharmacological therapeutic options, which vary in their effectiveness and adverse effect profiles, are available. Sustained increase in platelet count is seen with splenectomy and splenic artery embolization, in contrast to only transient rise with platelet transfusion. However, their routine use is limited by complications. Different thrombopoietin analogues have been tried. The use of synthetic thrombopoietins, such as recombinant human TPO and pegylated recombinant human megakaryocyte growth and development factor (PEG-rHuMDGF), has been hampered by the development of neutralizing antibodies. Thrombopoietin-mimetic agents, in particular, eltrombopag and romiplostim, have been shown to be safe and effective for HCV-related thrombocytopenia in various studies, and they increase platelet count without eliciting any immunogenicity Other treatment modalities including newer TPO analogues-AMG-51, PEG-TPOmp and AKR-501, recombinant human IL-11 (rhIL-11, Oprelvekin), recombinant human erythropoietin (rhEPO), danazol and L-carnitine have shown promising early result with improving thrombocytopenia. Thrombocytopenia in chronic HCV infection remain a major problem, however the recent change in DAAs without IFN, as the frontline therapy for HCV, permit to avoid the dilemmas associated with initiating or maintaining IFN based anti-viral therapy.

A Pilot Study of Eltrombopag Plus G-CSF for Human CD34+ Cell Mobilization in Patients with Multiple Myeloma Undergoing Autologous Stem Cell Transplant

Background: Administration of recombinant human thrombopoietin (rhTPO) with G-CSF for stem cell mobilization is associated with high CD34+ stem cell yield, rapid neutrophil recovery following autologus transplantation (ASCT), and decreased red blood cell (rbc) and platelet (plt) transfusions (Solomo, G. et al. Blood 1999). However, clinical development of rhTPO was complicated by the formation of neutralizing anti-TPO antibodies (Li, J. et al. Blood 2001), prompting discontinuation of further clinical development of recombinant TPO. Eltrombopag (Elt) is an orally bioavailable small molecule thrombopoietin receptor (TPO-R) agonist approved by the FDA for treatment of chronic immune thrombocytopenic purpura (ITP). In vitro studies have demonstrated that Elt promotes megakaryocyte proliferation and differentiation of CD34+ bone marrow progenitor cells (Erickson-Miller CL Stem Cells 2009), suggesting that Elt might be a surrogate for rhTPO for stem cell mobilization. In this pilot trial, we evaluated the combination of Elt plus standard G-CSF and cyclophosphamide (C) for stem cell mobilization in patients (pts) with multiple myeloma (MM), a disease for which ASCT remains a standard of care (Blade et al. Blood 2010).Methods: Primary objectives included determination of the median number of CD34+ cells/kg mobilized and the maximum tolerated dose (MTD) of Elt. Pts had MM that was stable or responsive to at least two cycles of chemotherapy with plans for stem cell mobilization and ASCT. Four pts were to be enrolled in each of four dose escalation arms in which they received 0 (Arm D), 50 (Arm A), 100 (Arm B), or 150 mg (Arm C) of eltrombopag in combination with standard C + G-CSF. Adverse events (AEs) were graded by NCI-CTCAE v4.Results: 17 pts have been screened and enrolled to date. Two patients withdrew consent prior to receiving Elt and were excluded from statistical analysis. 15 patients have completed participation in the study to date and two patients remain to be enrolled in Arm C. The first subject in Arm A experienced delayed engraftment that was determined to be unrelated to ELT; rather, the event was attributed to administration of a one-time high dose of corticosteroid for management of a severe hypersensitivity reaction to DMSO that occurred during stem cell infusion. A second subject in Arm A had undergone mobilization with Elt prior to the previously described delayed engraftment event, and to ensure safety underwent a second mobilization with G-CSF and plerixafor. During ASCT, this patient received cells from the second mobilization procedure. While neither event met criteria for a dose-limiting toxicity, the protocol was amended such that three additional patients enrolled in Arm A underwent two rounds of mobilization - the first with Elt plus C and G-CSF and a second with G-CSF plus plerixafor - and received as part of ASCT cells mobilized with Elt. Each of these patients engrafted successfully. The median number of CD34+ cells/kg collected during the first collection day of apheresis in Arms D, A, B, and C was 8.0, 11.0, 15.3, and 26.4. The median total number of CD34+ stem cells collected following mobilization with Elt plus C and G-CSF in Arms D, A, B, and C was 13.2, 12.7, 15.4, and 26.4. The percentage of patients in Arms D, A, B, and C who achieved a target collection of 8 x 10^6 CD34+ stem cells in one collection day was 50, 60, 75, and 100%. There have been no severe adverse events related to Elt .Conclusions: Administration of Elt in combination with C plus G-CSF for stem cell mobilization in pts with MM undergoing ASCT was safe and well tolerated, with no DLTs or severe AEs attributable to Elt. The small size of this pilot study precludes formal statistical comparison of outcomes across treatment Arms, but there appears to be a trend toward increase in yield of CD34+ cells and decrease in apheresis procedures required with increasing doses of Elt. DisclosuresRichardson:Jazz Pharmaceuticals: Consultancy, Membership on an entity's Board of Directors or advisory committees.

Effects of Anti-Glycoprotein Antibodies on Response of Immune Thrombocytopenia Patients to Thrombopoietin Receptor Agonists and on Megakaryocytes Viability

BackgroundImmune Thrombocytopenia (ITP) is a bleeding disorder due to a combination of increased platelet destruction and reduced production, often secondary to anti-platelet/megakaryocyte antibodies.The presence of antibodies to glycoproteins (GP) IIb/IIIa (integrin αIIbβ3) and GPIb/IX, detected in majority of ITP patients, may correspond to different responses to treatment, i.e., anti-GPIb is associated with more severe disease, and less responsive to intravenous immunoglobulins and steroids.Thrombopoietin Receptor Agonists (TPO-RA) increase platelet production by stimulation of megakaryopoesis. Predictors of response to TPO-RA and influence of antibody profile on response are currently unknown. In our previous study we investigated Absolute Immature Platelet Fraction (A-IPF) prior to TPO-RA treatment and did not find a correlation between A-IPF, anti-GP antibodies, and platelet counts.The aims of this study were to further investigate:1. The role of anti-GP antibodies in response to TPO-RA;2. Effect of patients' antibodies on megakaryocyte (MK) viability, maturation, apoptosis and formation of proplatelets (in vitro);3. The influence of patients' clinical characteristics on response to TPO-RA.Materials and Methods91 patients with persistent or chronic ITP, were treated at Weill Medical College of Cornell University until January 2015 with TPO-RAs: 52 patients received eltrombopag, 22 romiplostim and 17 avatrombopag. Serum samples of 84 patients were analyzed for the presence of anti-GP by MAIPA assay as previously described.Patients with baseline platelet counts less than <30x109/L were defined as responders to TPO-RA if the average of their six median monthly platelet counts was ≥50x109/L and doubled from average baseline counts (prior to TPO-RA). Patients with baseline platelet counts 30-50x109/L were responders if the average platelet count was ≥75x109/L.MKs were derived from human umbilical cord blood stem cells as previously described. Cells were grown using SFEM medium, adding on day 0 of culture 50 ng/ml recombinant TPO and aliquots of serum of ITP patients or healthy controls. The percentages of immature (CD41+/CD42-), mature (CD41+/CD42+), viable and apoptotic MKs were analyzed by flow cytometry on day 12. Apoptosis was analyzed by measuring Mitochondrial Outer Membane Potential (MOMP) and Phosphatidyl Serine (PS) externalization. MKs were considered apoptotic if they had positive staining for PS externalization, viable if positive for MOMP, and dead if positive for 7-Aminoactinomycin D (7AAD).Proplatelet formation by MKs was analyzed by microscopy.Statistical analysis using unpaired T-test and Pearson correlation test were performed.ResultsNinety-one patients were included, 40 male (44%) and 51 female (56%), with a median age of 37.4 years (range 2-87). Median duration of ITP before TPO-RA treatment was 8 years (range 0.3-45).The 18/91 (19.8%) non-responders to TPO-RA were not different from the 73/91 responders in age, gender, number of prior treatments, duration of ITP, and past splenectomy. The presence of either or both anti-GP antibodies was correlated with average lower platelet counts on TPO-RA: 82.3 x109/L versus 123x109/L in patients without detected antibodies ("neither") (p=0.003). However, the response to TPO-RA was not influenced by the type of antibody: in patients with anti-GPIb the average platelet count was 76.1x109/L, and with anti-GPIIb/IIIa 80.7x109/L (Figure 1). In culture, excess dead MKs were found in anti-GPIb group and antiGPIb&antiGPIIb/IIIa group compared to "neither" group (p=0.0013 and p=0.027 respectively) and comparing antiGPIb&antiGPIIb/IIIa to control (p=0.0025). We did not observe changes in the degree of MK apoptisis or in MK maturation in the presence of serum antibodies. In cultures treated with serum of patients having anti-GPIb, less proplatelets were detected comparing to control (p=0.044) or to "neither" (p=0.0039).We conclude that patients with anti-GP antibodies respond less to TPO-RA, however there is no difference in response to TPO-RA between patients having anti-GPIb and anti-GPIIb/IIIa, unlike responses to other treatment modalities (e.g., steroids or immunoglobulins). TPO-RA could be a preferable treatment option in ITP patients having anti-GPIb. [Display omitted] DisclosuresOff Label Use: Eltrombopag, romiplostim and avatrombopag are a thrombopoietin receptor agonist approved for the treatment of thrombocytopenia in adults with chronic ITP. In some preliminary studies these medicines found as safe and effective treatment option in children and adolescents. Bussel:amgen: Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Research Funding; GlaxoSmithKline: Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Research Funding; Cangene: Research Funding; Novartis: Consultancy, Research Funding; Genzyme: Consultancy; BiologicTx: Research Funding; Ligand: Consultancy, Research Funding; Eisai: Consultancy, Research Funding; Shionogi: Consultancy, Research Funding; momenta: Consultancy; Protalex: Consultancy; Symphogen: Consultancy.

Recombinant human thrombopoietin promotes hematopoietic reconstruction after severe whole body irradiation.

Recombinant human thrombopoietin (rHuTPO) is a drug that is used clinically to promote megakaryocyte and platelet generation. Here, we report the mitigative effect of rHuTPO (administered after exposure) against severe whole body irradiation in mice. Injection of rHuTPO for 14 consecutive days following exposure significantly improved the survival rate of lethally irradiated mice. RHuTPO treatment notably increased bone marrow cell density and LSK cell numbers in the mice after sub-lethal irradiation primarily by promoting residual HSC proliferation. In lethally irradiated mice with hematopoietic cell transplantation, rHuTPO treatment increased the survival rate and enhanced hematopoietic cell engraftment compared with the placebo treatment. Our observations indicate that recombinant human TPO might have a therapeutic role in promoting hematopoietic reconstitution and HSC engraftment.

Long-term outcome of HLA-haploidentical hematopoietic SCT without in vitro T-cell depletion for adult severe aplastic anemia after modified conditioning and supportive therapy

HLA-haploidentical hematopoietic SCT (HSCT) is an option for severe aplastic anemia (SAA) patients. Here, we evaluated the outcomes of 26 adult-SAA patients who received HLA-haploidentical HSCT in five transplant centers in southwestern China. Most of the patients in this study failed prior therapy and were transfused heavily before the transplantation. The patients received fludarabine+cyclophosphamide+antithymocyte globulin as conditioning regimens and then unmanipulated peripheral blood plus marrow transplantation. Micafungin, i.v. Ig and recombinant human TPO were used for post-grafting infection prevention and supportive care. Of 26 patients, 25 achieved engraftment at a median of 13 days (range, 11-19 days) after HSCT. One of 25 patients experienced graft rejection and did not achieve sustained engraftment after second HSCT. Therefore, the final engraftment rate was 92.3%. Three of 25 (12%) patients developed acute GVHD, 10 of 25 (40%) patients developed chronic GVHD (9 with limited whereas the other with extensive). The OS rate was 84.6% and the average follow-up time was 1313.2 (738-2005) days for surviving patients. This encouraging result suggests that HLA-haploidentical HSCT is an effective therapeutic option for adults with acquired SAA if an HLA-identical donor is not available.

Does thyroid peroxidase provide an antigenic link between thyroid autoimmunity and breast cancer?

Women with breast cancer (BC) and antithyroid peroxidase (TPO) autoantibodies (TPOAb) have a better prognosis than women lacking TPOAb. Sera from women with TPOAb displayed immunoreactivity to BC tissue by immunofluorescence that was not apparent in women without TPOAb. We hypothesize a BC/thyroid shared antigen that provides a target for humoral or cell-mediated immune activity; candidates include the sodium/iodide symporter (expressed in thyroid and BC), cross-reacting epitopes in TPO and lactoperoxidase (LPO) or TPO itself. As the association is with TPOAb, we investigated TPO expression in BC, breast peritumoral tissue (PT), other tissues (tumoral and not) and thyroid as positive control. Transcripts for known and novel TPO isoforms were detected in BC (n = 8) and PT (n = 8) but at approximately 10(4) -fold lower than in thyroid while in non-BC tumors (n = 5) they were at the limit of detection. TPO was expressed also in adipose tissue (n = 17), 10(3) -fold lower than in thyroid. Full length TPO (Mr 105-110 kDa) was detected in Western blots in the majority of examined tissues; preabsorption of the TPO antibody with recombinant TPO (but not LPO) reduced the signal, indicating specificity. The same occurred with some lower molecular weight bands, which could correspond to smaller TPO transcript isoforms, present in all samples. In conclusion, TPO is weakly expressed in BC and other tissues; this could partly explain the high frequency and protective role of TPOAb in BC patients. Further studies will investigate tissue specificity, function and immunogenicity of the novel TPO variants (some BC-specific) identified.

Recombinant Human Thrombopoietin Alleviates Infection-Associated Thrombocytopenia

To examine the efficacy and adverse events of recombinant human thrombopoietin (rhTPO) in the treatment of infection-associated thrombocytopenia in senile patients. The current study is a retrospective analysis of the patients receiving rhTPO for infection-associated thrombocytopenia in our hospital. Forty-nine cases were included in the analysis as rhTPO group. The absolute platelet count after treatment, increase in platelet count, and the overall response rate were considerably higher in the rhTPO group than that in the control group. Improvement in bleeding score was higher in the rhTPO treatment group than that in the control group (2.1 ± 5.4 vs 0.4 ± 1.7). Bleeding event was stopped in 68.2% of the patients after rhTPO treatment and in 35% of the patients in the control group (P = .032). A stratified analysis indicated that the therapeutic efficacy is much better in patients without organ failure. Recombinant human TPO is effective in alleviating infection-associated thrombocytopenia and hemorrhage in senile patients, particularly if given prior to the emergence of organ failure.

Aberrant Megakaryocyte Gene Expression Contributes to Primary Myelofibrosis.

AbstractAbstract 2867▪▪This icon denotes a clinically relevant abstractPrimary myelofibrosis (PMF) is a clonal hematologic malignancy, which results from the transformation of a pluripotent hematopoietic progenitor cell. A major consequence of this transformation is increased hematopoiesis and an overproduction of abnormal blood cells. PMF is associated with bone marrow fibrosis, extramedullary hematopoiesis, increased numbers of circulating CD34+ cells, splenomegaly, and a propensity to evolve to AML. Patients also display anemia and thrombocytopenia and harbor abnormal, immature megakaryocytes (Mks) in their bone marrow and spleen. PMF patients can present well known mutations including JAK2V617F (65%), MPL (10%), TET2 (17%), CBL (6%), IDH (4%,), which are not specific to the disease and are also present in polycythemia vera, essential thrombocythemia and AML.We hypothesize that the genetic events associated with PMF, including MPL and JAK2 mutations, contribute to defects in Mk maturation, but that additional changes are needed to explain the striking abnormalities seen in PMF relative to the other myeloproliferative diseases. Although there have been studies to examine the aberrant gene expression program of CD34+ cells of PMF patients, we chose to examine the changes that occur in gene expression specifically in Mks as a way to better understand their abnormal differentiation and to determine their contribution to the disease.Primary CD34+ cells from PMF patients and healthy donors were cultivated in serum free media supplemented with recombinant TPO, BSA, liposomes, insulin and transferrin to support the growth of Mks. After 10 days of differentiation, we evaluated the cultures for proliferation, apoptosis and differentiation by flow cytometry. We found that PMF specimens gave rise to a lower percentage of mature (CD41+CD42+) cells as compared to healthy donors, but showed, a lower ploidy level, a greater proliferation and increased survival. These observations are consistent with the clinical observations that PMF bone marrow is characterized by an increased number of immature, dysplastic Mks.We used flow cytometry to collect two populations of cells for analysis: immature CD41+CD42− Mks, and CD41+CD42+ mature MKs. After sorting, we extracted RNA and performed whole genome microarray analysis with Illumina Human HT12-v4 arrays on cohorts of PMF and control specimens. Gene expression data were analyzed by GeneSpring and Gene Set Enrichment Analysis (GSEA). We found that the CD41+CD42− MKs derived from PMF progenitors showed reduced expression of GATA1 as compared to control cells, as expected based on previous study by Dr. Alessandro Vannuchi.GeneSpring analysis revealed that myeloid transcription factors, including CEBPa, GFI1, and SPI1 (PU.1), which are not expressed in normal MKs, are strikingly and significantly overexpressed in PMF samples. Moreover, c-myb, which regulates the erythroid/Mk cell fate decision, FOG-1 and AML1, are also overexpressed in PMF Mks. This aberrant myeloid gene expression program in PMF Mks is reminiscent of a similar defect we observed in Mks with reduced expression of GATA-1 and GATA-2. We predict that reduced levels of GATA-1 protein in PMF Mks, as reported by Dr. Alessandro Vannucchi and colleagues, is in part responsible for the aberrant growth and differentiation of the PMF Mks. Our data support the model that PMF Mks are defective in their ability to properly regulate expression of hematopoietic regulators. Further analysis by GSEA revealed that hematopoietic and cytokine pathways are among those that are highly enriched in PMF Mks.We recently reported that the molecules dimethylfasudil (diMF) and MLN9237 are able to selectively increase ploidy, Mk surface marker expression, and apoptosis of malignant Mks. We treated Mks derived from PMF progenitor cells with diMF and observed a high increase in polyploidization accompanied with a reduction of Mks proliferation. Thus, diMF is able to partially restore Mk differentiation of PMF cells, supporting the testing of polyploidy inducers in myelofibrosis patients. Disclosures:Pardanani:Sanofi-Aventis: Clinical trial support Other; YM BioSciences: Clinical trial support, Clinical trial support Other; Bristol-Myers Squibb: Clinical trial support, Clinical trial support Other.

Analysis of gene expression profiles of megakaryocytes from human cord blood CD34+ cells in vitro expanded using DNA microarray

Objective To study the gene expression profiles of megakaryocytes(MKs) from human cord blood CD34+ cells in vitro expanded and to understand megakaryopoiesis at the molecular level. Methods CD34+ cells were isolated using density gradient centrifugation and magnetic activated cell sorting. The cells were cultured and stimulated with recombinant human TPO ( 100 ng/ml). After 12 days, the MKs fraction was separated using an anti-CD41 monoclonal antibody by immunomagnetic sorting. The gene expression profiles of MKs, non-MKs as well as meg-01 cells were studied by gene chip assay. THBSI, HOX A9,β-actin, lL-8,Annexin A6, FGF-8 were selected to validate the gene chip results by RT-PCR. Results A total of 116 genes between MKs and non-MKs cells were significantly different, 52 genes were up-regulated and 64 genes were down-regulated. In addition, 158 genes between MKs and meg-01 cells were significantly different, 71 genes were up-regulated and 87 genes were down-regulated. THBSI showed higher expression in MKs than in non-MKs. HOXA9 showed lower expression in MKs than in non-MKs. The expression of β-actin did not show any significant difference in MKs and non-MKs. IL-8 showed higher expression in MKs than in meg-01 cells, while ANXA6 showed lower expression in MKs than in meg-01 cells. The expression of FGF-8 did not show any significant difference between MKs and meg-01 cells. Conclusions MKs, non-MKs and meg-01 cells show different gene expression profiles. The regulatory genes include stress response genes,immune related genes, DNA synthesis and repair genes, metabolism genes, pro-onco genes and tumor suppressor genes. Key words: Megakaryocytes; Gene expression; Oligonucleotide array sequence analysis; In vitro

Biology and Chemistry of Thrombopoietic Agents

Endogenous thrombopoietin (eTPO) regulates platelet production by increasing the number, ploidy, and maturation rate of bone marrow megakaryocytes. Early attempts to treat thrombocytopenia by the administration of recombinant TPO were successful but were complicated by the development of antibodies to one of the recombinant proteins. Two new TPO mimetics have recently been approved by the US Food and Drug Administration (FDA) for the treatment of immune thrombocytopenia (ITP). Romiplostim is a peptide TPO mimetic composed of an IgG Fc fragment to which are attached four 14-amino acid TPO peptides that activate the TPO receptor by binding to the extracytoplasmic domain just like eTPO. Romiplostim is administered as a weekly subcutaneous injection. Eltrombopag, a nonpeptide TPO mimetic, is a 442-d drug that binds to a transmembrane site on the TPO receptor and thereby activates it. It is administered daily as an oral tablet. Administration of both romiplostim and eltrombopag to healthy volunteers produced a dose-dependent rise in platelet count beginning on day 5 and peaking at days 12 to 15. Both have been highly effective in increasing the platelet count in patients with ITP and are currently being studied in the treatment of other thrombocytopenic conditions (myelodysplastic syndrome, chemotherapy, liver disease).

Effectiveness of a Novel Humanized VB22B Minibody against Human TPO Receptor Compared with Recombinant TPO and Eltrombopag Using Primary Human CD34+ Cells and Platelets.

Abstract 4559Thrombopoietin (TPO) is a cytokine produced primarily by the liver and kidney that regulates platelet production by stimulating proliferation and differentiation of hematopoietic stem cells, megakaryocytic progenitor cells and megakaryocytes via activation of its receptor, c-Mpl. Recently, TPO receptor agonists such as eltrombopag and romiplostim have been approved for chronic ITP. huVB22B was created as a novel humanized form of murine sc(Fv) 2VB22B minibody (BLOOD, 2005) which activates human c-Mpl by CDR grafting. The advent of these various TPO receptor agonists prompted us to consider the differences in their mechanisms of action, efficacy or potency. However, to date, there has been no in vivo or in vitro study directly comparing the effects of different TPO receptor agonists. In this study, we compared the efficacy of huVB22B on CFU-GM, CFU-E, CFU-Megakaryocyte (CFU-MK), megakaryocyte maturation (DNA ploidy and proplatelet formation) with those of recombinant human TPO (rhTPO) and eltrombopag. Primary human CD34+ bone marrow cells were cultured with various concentrations of rhTPO, huVB22B and eltrombopag using methylcellulose based media. In serum-free condition, 0.286 nM rhTPO, 0.182 nM huVB22B and 17.7 mcM eltrombopag demonstrated almost equivalent efficacy of megakaryocyte colony formation. At these concentrations, all agents demonstrated similar in vitro efficacy for colony formation of CFU-GM and CFU-E, proplatelet formation and nuclear maturation of megakaryocytes. In preliminary results, huVB22B induced maturation of CFU-MK earlier than rhTPO and eltrombopag, suggesting that huVB22B might have some potential to increase human platelets faster than other agents in vivo. This is compatible with the observation that huVB22B induced tyrosine phosphorylation of STAT3, STAT5 and JAK2 faster and stronger than rhTPO and eltrombopag in human primary platelets. Both rhTPO and huVB22B enhanced low-dose ADP and collagen-induced human platelet aggregation in vitro. In contrast, eltrombopag did not enhance ADP or collagen-induced platelet aggregation, although it induced activation of JAK-STAT pathway in human platelets. Contrary to the fact that huVB22B induces phosphorylation of intracellular signaling molecules faster and stronger than rhTPO in human platelets, the priming effect by huVB22B on platelet aggregation was much weaker than rhTPO. In conclusion, we confirmed that newly created huVB22B minibody induced colony formation of CFU-MK, CFU-E, CFU-GM and maturation of megakaryocytes from human bone marrow-derived CD34+ cells in vitro. The differences among TPO receptor agonists observed in our study would lead to further understanding of the basic biology of megakaryopoiesis and the action of TPO receptor agonists. Disclosures:Okamoto:Alexion: Research Funding. Miyakawa:GlaxoSmithKline: Consultancy.

Comparative analyses of the small molecule thrombopoietin receptor agonist eltrombopag and thrombopoietin on in vitro platelet function

The thrombopoietin receptor (TPOR) is a therapeutic target for treatment of thrombocytopenia because stimulation of this receptor results in enhanced megakaryocyte proliferation, differentiation, and ultimately platelet production. In addition to effects on megakaryocytes, TPOR stimulation also impacts platelet function. The present study examined platelet function following stimulation with the small molecule TPOR agonist eltrombopag. Platelets were obtained from healthy volunteers, and signal transduction pathway activation was examined in washed platelet preparations. Platelet aggregation was examined in both washed platelet preparations and platelet-rich plasma. Platelet alpha-granule release was determined via fluorescein-activated cell sorting measurement of CD62P. In signal transduction studies of washed human platelets, eltrombopag induced the phosphorylation signal transducers and activators of transcription (STAT) proteins with no phosphorylation of Akt, whereas recombinant human TPO (rhTPO) induced the phosphorylation of Akt as well as STAT-1, -3, and -5. In studies conducted at subthreshold/submaximal concentrations of adenosine diphosphate (ADP) or collagen, eltrombopag pretreatment did not result in platelet aggregation. In contrast, rhTPO acted in synergy with submaximal concentrations of ADP or collagen to induce maximal aggregation under all conditions examined. Similarly, platelet activation as examined via surface expression of CD62P was not enhanced by eltrombopag pretreatment as compared to rhTPO. These results demonstrate that the nonpeptidyl TPOR agonist eltrombopag stimulates platelet signal transduction with little or no effect on overall platelet function, in contrast to TPO, which significantly primes platelet activation. These data demonstrate that effects of TPOR ligands on platelet function can vary depending on the specific mechanism utilized to stimulate the TPOR.

A Nonpeptidyl, Hydrazone Class, Thrombopoietin Receptor Agonist (TpoRA), SB559457, Selectively Kills Primary Human Myeloid Leukemia Cells

Treatment of thrombocytopenia, whether autoimmune, secondary to marrow failure, or iatrogenic, can become problematic. Stimulation of endogenous megakaryocyte (MEG) development might be an effective strategy and it was originally hoped that this could be accomplished with thrombopoietin (Tpo), the natural ligand of the thrombopoietin receptor (TpoR) and regulator of MEG development. When formulated Tpo proved immunogenic, development of small molecule TpoR agonists (TpoRA) was undertaken. In course of investigating the biological effects, and mechanism of action, of SB559457 (a nonpeptidyl, hydrazone class, TpoRA supplied by GlaxoSmithKline (GSK), Collegeville, PA) we found that the molecule robustly stimulated human megakaryocytopoiesis in vitro. In anticipation of TpoRA use in the treatment of patients with hematologic malignancies, we also evaluated SB559457's effect on human leukemia cell (HLC) growth in vitro since it is known that HLCs may express TpoR. Specifically, we examined the effects of SB559457 on samples obtained from 21 patients with AML, and 7 with ALL. Surprisingly, SB559457 not only failed to stimulate HLC growth, it proved toxic to primary acute myelogenous leukemia (AML). In 20 of 21 primary AML samples exposed to SB559457 (5μM) a large fraction (70–90%) of cells died between days 3 and 6 of culture, while untreated control cells survived. No significant effects on cell growth or viability were observed in the ALL patient samples. To investigate the mechanism(s) of AML cell killing, we examined signaling cascades initiated by SB559457 compared to recombinant human Tpo (rhTpo), which had no apparent effect on AML cell growth. Initially, we employed N2C-Tpo cells (a Tpo dependent megakaryoblastic cell line that express TpoR) and focused on kinases known to be phosphorylated after TpoR activation; STAT5, ERK, p70S6, and ribosomal kinase S6. When N2C-Tpo cells were stimulated with rhTpo (2.8μM, 30 min) all these kinases were highly phosphorylated. In contrast, none were phosphorylated in SB559457 (5μM, 30min) stimulated cells. Normal human CD34+ marrow cells were next examined using the same experimental conditions. In contrast to N2C-Tpo cells, ERK, and p70S6 kinases were both phosphorylated after exposure to SB559457 but STAT5 remained unphosphorylated. Since AML cells may require STAT5 activation for growth, we hypothesized that perturbation of STAT5 activation might be involved in the apoptotic mechanism. The molecular consequences of differential signaling were then pursued by Affymetrix GeneChip analysis. Remarkably, in 5 separate primary AML cell samples stimulated for 6 hours with either Tpo or SB559457 we found a statistically significant difference in expression in only 2 of 22000 genes represented on the chips: GAPDH and Redd1. Both of these genes are induced in stressed cells undergoing apoptosis. In addition, stimulation of primary AML cells with SB559457 resulted in increased phosphorylation of p70S6/S6 kinases, both downstream targets of mTOR kinase. Accordingly, we hypothesize that SB559457 mediated activation of mTOR pathway leads to a stress response in primary myeloid leukemia cells as reflected by the increased expression of Redd1 and GAPDH. Further investigation of this pathway, and the leukemic cell response to SB559457 exposure may lead to development of novel strategies for the treatment of myeloid leukemias.

Revisiting therapeutic strategies in radiation casualties

Nuclear/radiological threats have evolved and scenarios for terrorist attacks involving radioactive material have been identified as complex situations. Mass casualty scenarios may happen, and individuals may be exposed to intentionally hidden sources of high activity, resulting in delayed diagnosis and treatment of acute radiation syndrome (ARS). Moreover, ARS must be considered as an emergency in order to better anticipate delayed radiation toxicity. In this context, therapeutic strategies in radiation casualties have to be revisited and new pharmacological approaches developed. B6D2F1 mice were total-body irradiated (TBI) with a 9 Gy gamma dose and then received intraperitoneal doses of either early (stem cell factor + FLT-3 ligand + thrombopoietin + interleukin-3 [SFT3] +/- keratinocyte growth factor (KGF); stem cell factor + erythropoietin + Peg-filgrastim [SEG]) or delayed treatments (SFT3 +/- KGF, erythropoietin, or hyaluronic acid). Survival was monitored and bone marrow hematopoiesis evaluated at 300 days following early treatments. SFT3 anti-apoptotic cytokine combination administered early (2 hours and 24 hours) after lethal TBI induced 60% survival versus 5% in controls. Early SEG treatment may be an alternative to SFT3 in terms of survival (55%), but SEG benefit might be obtained at the expense of long-term hematopoiesis. SFT3 + KGF induced 75% survival. No effectiveness was observed, over antimicrobial supportive care, when administration of SFT3 or its tested combinations was delayed at 48 hours. As a potentially multi-organ failure, ARS requires global therapy, beyond the hematopoietic syndrome, which may include pleiotropic cytokines such as KGF.

Differential effects of recombinant thrombopoietin and bone marrow stromal-conditioned media on neonatal versus adult megakaryocytes

AbstractUmbilical cord blood (CB) is a valuable source of stem cells for transplantation, but CB transplantations are frequently complicated by delayed platelet engraftment. The reasons underlying this are unclear. We hypothesized that CB- and peripheral-blood (PB)–derived megakaryocytes (MKs) respond differently to the adult hematopoietic microenvironment and to thrombopoietin (Tpo). To test this, we cultured CB- and PB-CD34+ cells in adult bone marrow stromal conditioned media (CM) or unconditioned media (UCM) with increasing concentrations of recombinant Tpo and compared the effects of these conditions on CB-versus PB-MKs. PB-MKs reached highest ploidy in response to UCM + 100 ng/mL rTpo, and the addition of CM inhibited their maturation. In contrast, CB-MKs reached highest ploidy in CM without rTpo, and high rTpo concentrations (> 0.1 ng/mL) inhibited their maturation. This is the first evidence that human neonatal and adult MKs have substantially different biologic responses to Tpo and potentially to other cytokines.

Effects of SB559457, a Novel Small Molecule Thrombopoietin Receptor (TpoR) Agonist, on Human Hematopoietic Cell Growth and Differentiation.

Treatment of thrombocytopenia resulting from chemotherapy, radiation, or stem cell transplantation remains problematic. Hopes that TPO, the major regulator of megakaryocyte maturation, might prove clinically useful for this purpose have not been fulfilled, in part because of untoward reactions to the molecule. To avoid undesirable side effects, small molecule TpoR agonists are being developed. We have evaluated the biological effects of one such molecule, SB559457, a hydrazone compound, on hematopoietic cells. We first examined SB559457's ability to stimulate proliferation of the recombinant human TPO (rhTPO) dependent cell line UT-7/TPO. We found a dose dependent augmentation of cell proliferation at doses between 1 and 10 μM, with maximal effects, compared to 100ng/ml of rhTPO at 10μM. We then evaluated SB559457's growth promoting effects on normal human CD34+ cells. We again found growth stimulation to be dose dependent between 1 and 10 μM and also noted enhancement when SB559457 was combined with IL-3 and SCF. Specifically, incubation of CD34 cells in 10 μM SB559457 for 7 days lead to a 2 fold increase in cell number; incubation of cells with IL3 and SCF in addition to SB559457 lead to a 12 fold increase in cell number after 7 days. Both results were comparable to those seen with 100 ng/ml of rhTPO. Curiously, the addition of IL-6 to cultures containing IL-3 and SCF compromised the ability of SB559457, but not Tpo, to stimulate CD34+ cell growth. To determine if SB559457 stimulates megakaryocyte differentiation directly, CD34+ cells were incubated in cytokines with rhTPO or SB559457 for periods of 7–10 days, after which time cells in culture were examined for degree of polyploidization, and expression of megakaryocyte lineage markers CD41 and CD61. 14% of cells grown in SB559457 showed greater than 4N DNA content compared to 8% in rhTPO. These same cells expressed the megakaryocyte markers CD41 and CD61on 35% of cells, comparable to the results with rhTPO. Preliminary results demonstrate that incubation of CD34 cells in SB559457 maintained stem cells numbers, as assayed by NOD/SCID engraftment, for at least 4 days at a level comparable to the levels maintained by rhTPO. These results demonstrate that SB559457 is a bona fide Tpo R agonist that likely acts through activation of c-mpl, the receptor for TPO. These experiments provide a rationale for the evaluation of SB559457, or SB559457 congeners, in patients with thrombocytopenia.

SB 497115-GR, a Low Molecular Weight TPOR Agonist, Does Not Induce Platelet Activation or Enhance Agonist-Induced Platelet Aggregation in Vitro.

SB-497115-GR is a low molecular weight, orally active molecule that requires Tpo receptor (TpoR) expession for activity and activates JAK/STAT signalling in platelets. The ability of recombinant Tpo to enhance agonist-induced platelet aggregation is well-documented, thus this study was undertaken to compare the ability of rhTpo (150 ng/ml) and SB-497115 (0.01–10 uM) to affect platelet activation. Platelets were obtained from healthy human volunteers, following written informed consent, by standard venipuncture technique. Citrated whole blood was used directly in platelet activation and P-selectin assays within 15 minutes. Platelet aggregation was assessed by light transmittance aggregometry. Aggregation was induced by a submaximal concentration of adenosine diphosphate (ADP) (1.0 or 1.5 uM) in experiments examining synergy with rhTpo (150ng/mL) or SB-497115 (0.01–10 uM). For examination of potential inhibitory actions of SB-497115 on platelet function, aggregation was induced by ADP (3 uM), collagen (2 ug/mL), or the thrombin receptor activating peptide (TRAP; 20 uM). Pre-treatment of platelet samples with rhTpo, but not SB-497115, potentiated the effects of 1.0 or 1.5 uM ADP. No inhibitory effect on normal ADP, Collagen or TRAP-induced aggregation was seen with either SB-497115-GR or rhTpo. In experiments investigating the expression of platelet P-selectin, an early marker of platelet activation, platelets labeled with anti-CD62P antibody were analyzed by flow cytometry. Data was expressed as percent of platelets positive for CD62P within the defined platelet gate. Treatment with SB-497115 (0.1 – 10 uM) demonstrated no induction of platelet CD62P expression above background levels. In contrast, rhTpo produced a modest but significant and reproducible increase in the percent of platelets positive for CD62P. This study demonstrates that, in contrast to rhTpo, the orally active small molecular weight agonist of the Tpo receptor, SB-497115-GR, does not mimic the ability of rhTpo to enhance ADP-induced aggregation of platelets. SB-497115-GR also did not directly induce P-selection expression on human platelets. Finally, SB-497115 has no antagonistic effect on ADP-, TRAP-, or collagen-induced platelet aggregation. Thus, SB-497115 is differentiated from rhTpo with respect to platelet activation.