ALBERT

Description: Chronic myelogenous leukemia (CML) is a clonal myeloproliferative disease (MPD) initiated by p210-BCR-ABL–mediated transformation of hematopoietic stem cells (HSCs). Inhibition of the ABL kinase alone is not sufficient to eradicate leukemic stem cells (LSCs). We have previously shown that the deficiency of Rac2 GTPase signaling, but not Rac1, in p210-BCR-ABL–transduced hematopoietic cells prolonged survival of mice with MPD. Here we demonstrate that absence of Rac2 GTPase prolongs survival of HSC-initiated, inducible Scl/p210-BCR-ABL (Scl/p210) binary transgenic mice, it induces apoptosis, and, unlike in normal HSC and progenitor (HSC/P), impairs LSC and progenitor (LSC/P) proliferation in vivo. As a result, Rac2 deficiency causes functional exhaustion of the LSC pool in vivo. This defect is not due to impaired interaction with the hematopoietic microenvironment as reflected by its unaltered adhesion, migration, and homing to recipient organs. In summary, Rac2 deficiency exhausts the LSC pool in vivo through impairment of oncogene-induced proliferation and survival signals.

Description: The characterization and targeting of Philadelphia chromosome positive (Ph+) acute lymphoblastic leukemia (ALL)–initiating cells remains unresolved. Expression of the polycomb protein Bmi1 is up-regulated in patients with advanced stages of chronic myelogenous leukemia (CML). We report that Bmi1 transforms and reprograms CML B-lymphoid progenitors into stem cell leukemia (Scl) promoter-driven, self-renewing, leukemia-initiating cells to result in B-lymphoid leukemia (B-ALL) in vivo. In vitro, highly proliferating and serially replatable myeloid and lymphoid colony-forming cultures could be established from BCR-ABL and Bmi1 coexpressing progenitors. However, unlike in vivo expanded CML B-lymphoid progenitors, hematopoietic stem cells, or multipotent progenitors, coexpressing BCR-ABL and Bmi1 did not initiate or propagate leukemia in a limiting dilution assay. Inducible genetic attenuation of BCR-ABL reversed Bmi1-driven B-ALL development, which was accompanied by induction of apoptosis of leukemic B-lymphoid progenitors and by long-term animal survival, suggesting that BCR-ABL is required to maintain B-ALL and that BCR-ABL and Bmi1 cooperate toward blast transformation in vivo. Our data indicate that BCR-ABL targeting itself is required to eradicate Ph+/Bmi1+ B-ALL–initiating cells and confirm their addiction to BCR-ABL signaling.

Description: Connexin-43 (Cx43), a gap junction protein involved in control of cell proliferation, differentiation and migration, has been suggested to have a role in hematopoiesis. Cx43 is highly expressed in osteoblasts and osteogenic progenitors (OB/P). To elucidate the biologic function of Cx43 in the hematopoietic microenvironment (HM) and its influence in hematopoietic stem cell (HSC) activity, we studied the hematopoietic function in an in vivo model of constitutive deficiency of Cx43 in OB/P. The deficiency of Cx43 in OB/P cells does not impair the steady state hematopoiesis, but disrupts the directional trafficking of HSC/progenitors (Ps) between the bone marrow (BM) and peripheral blood (PB). OB/P Cx43 is a crucial positive regulator of transstromal migration and homing of both HSCs and progenitors in an irradiated microenvironment. However, OB/P Cx43 deficiency in nonmyeloablated animals does not result in a homing defect but induces increased endosteal lodging and decreased mobilization of HSC/Ps associated with proliferation and expansion of Cxcl12-secreting mesenchymal/osteolineage cells in the BM HM in vivo. Cx43 controls the cellular content of the BM osteogenic microenvironment and is required for homing of HSC/Ps in myeloablated animals.

Description: The molecular basis for the divergence of the erythroid (red blood cell) and megakaryocyte (platelet) lineages from a common bipotent MEP (megakaryocyte-erythroid progenitor) remains undefined. We now demonstrate that Rgs18 (regulator of G protein signaling 18), a GAP (GTPase activating protein) factor and a transcriptional gene target of the Gfi1b transcriptional repressor complex, likely arbitrates this critical lineage decision downstream of Gfi1b. Rgs18 was identified in a chromatin immunoprecipitation (ChIP on chip) screen for Gfi1b/LSD1/Rcor1 targets in erythroid cells. Accordingly, Rgs18 expression was found to be up-regulated in LSD1 inhibited, and Gfi1b deficient erythroid cells confirming repression of this gene by Gfi1b and its co-factors in this lineage. In contrast, Rgs18 expression was comparable in megakaryocytic cells derived from wild type and gfi1b-/-hematopoietic progenitors indicating Gfi1b independent expression of Rgs18 in these cells. Manipulation of Rgs18 expression produced opposite effects in the erythroid and megakaryocytic lineages. Rgs18 inhibition retarded megakaryocytic differentiation while its ectopic over-expression promoted differentiation at the expense of proliferation. The reverse was observed in erythroid cells where Rgs18 inhibition produced an enhancement of differentiation while over-expression impaired erythropoiesis. Since Rgs signaling regulates the activity of downstream MAPK pathways we determined the status of these pathways in Rgs18 manipulated cells. Inhibition of Rgs18 stimulated ERK phosphorylation in megakaryocytes but diminished it in erythroid cells. In contrast, Rgs18 inhibition in erythroid cells elevated p38MAPK protein and phosphorylation levels. The opposite effects of Rgs18 manipulation on MAPK signaling in erythroid versus megakaryocytic cells while intriguing are consistent with the changes in differentiation and proliferation observed in each lineage, respectively. Although Rgs18 manipulation produced opposite effects in erythroid and megakaryocytic cells, the level and activity of this factor correlated similarly with those of the mutually antagonistic transcription factors Fli1 (Friend leukemia integration [site] 1) and KLF1/EKLF (Kruppel like factor1) in both cell types. In both lineages, Rgs18 protein levels correlated directly with Fli1, and inversely with KLF1, message levels. Since Fli1 promotes megakaryocytic, and KLF1 erythroid, development; these results demonstrate that Rgs18 promotes the emergence of megakaryocytic cells from bipotent MEPs by modulating MAPK signaling and altering Fli1/KLF1 stoichiometries. Although it is unclear why Gfi1b mediated repression of Rgs18 is erythroid specific even though the former is expressed in both lineages, these results demonstrate that repression of Rgs18 by Gfi1b in fetal liver MEPs limits megakaryopoiesis and augments erythropoiesis. However following megakaryocytic commitment, robust Gfi1b independent expression of Rgs18 drives differentiation of this lineage while continued repression of Rgs18 by Gfi1b in erythroid cells ensures their proper maturation. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 4421 Factor VIIa (fVIIa) is one of the key proteins in the blood coagulation cascade. It activates factors IX and × on a negatively charged phospholipid surface in either a TF-dependent or TF-independent fashion (Silverberg et al, 1977; Bom et al, 1990). Monroe et al (1997) demonstrated that fVIIa binds to activated platelets independent of TF and partially restores thrombin generation in an in vitro model of hemophilia. Thus, it appears that interaction of fVIIa with platelet phospholipids plays an important role. We report that binding of 1,2-dihexanoyl-sn-glycero-3-phospholipids -L-serine (C6PS) and 1,2-dihexanoyl-sn-glycero-3-phosphoethanolamine (C6PE) to fVIIa causes changes in its activity as well as structure. Titration with C6PS led to changes in intrinsic fluorescence indicative of two or more binding sites for this lipid. Similar titrations with C6PE indicated that it probably binds to a single site on the protein. Experiments are underway to test this initial conclusion. Both lipids bind with comparable affinity (kd ~ 165 and 160 μ M) when data were analyzed using a single site model. We also examined the effect of the soluble lipids on the activity of fVIIa. Both C6PS and C6PE binding increased fVIIa proteolytic and amidolytic activity, with the effect of C6PS being more pronounced. Based on current data, it appears that both lipids bind to a single weak site, but that binding of either to this site promotes binding of C6PS to a second, tighter, and C6PS-specific site, which seems to be crucial in regulating activity. Further experiments are underway to test this hypothesis. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 113 The Kruppel-like factor 5 (Klf5) is a transcription factor that regulates cellular signaling and is involved in cell proliferation and oncogenesis. Recent studies have implicated Klf5 in the self-renewal and maintenance of pluripotent stem cells. In this project, we examined the role of Klf5 in hematopoietic stem/progenitor cells (HSC/P) activity. Using a murine model of inducible deficiency of Klf5 in hematopoietic tissue (Mx1-Cre/Klf5flox/flox, Klf5-deficient), we observed a severe engraftment deficiency in competitive repopulation assays in primary and secondary recipients, although it did not cause any significant hematopoietic defects basally or after chemotherapy stress in primary mice. Engraftment failure of Klf5-deficient HSC was associated with a severe defect in homing (1.8±0.8% vs 6.0±2.0% for Klf5-deficient and WT HSC/P, respectively, p

Description: Introduction The increased risk of thromboembolic events under low oxygen environments such as high altitude regions is a well known phenomenon but with little knowledge about molecular events underlying its pathogenesis. We have recently reported the proteomic changes in hyperreactive platelets under hypoxia and demonstrated that increased activity of protease calpain is crucial for the induction of prothrombotic phenotype under hypoxic environment (Tyagi et al, 2014). However, considering the complex nature of hemostatic reactions, multiple aspects of the regulation of hemostatic balance under hypoxia need to be understood. Hypoxic environment creates oxidative stress in biological system which needs to be contained by counteractive factors. One of such factor, the Protein disulfide isomerase (PDI), which catalyzes the formation and rearrangements of disulfide bonds on proteins, has been found to be upregulated in multiple cell types and is believed to be protective under oxidative stress. The extracellular PDI has been recently reported to play a key role in initial thrombotic events in vivo and in vitro, but whether it affects the hemostasis under hypoxia remains unknown. We analyzed the role played by PDI in calpain regulated hypoxia induced prothrombotic phenotype by using specific PDI inhibitor Quercetin-3-rutinoside (Q-3R) in an animal model. Method:The Sprague-Dawley male rats were exposed to simulated hypobaric hypoxia in a specially designed animal decompression chamber maintained at pressure of 366 torr (equivalent to altitude of 6096m) for 3h duration. The exposed group of animals was infused with either Q-3R or vehicle, via tail vein injection prior to hypoxia exposure. The effect of PDI inhibitor on coagulation was analyzed by measuring clotting time and Prothrombin time. The PDI activity was measured in plasma by insulin reduction assay. The plasma calpain activity was measured by fluorescence based assay. The status of oxidative damage was assessed by measuring MDA levels which are hallmark of oxidative stress. We also analyzed the factor V activity in plasma of animals by standard clot based assay using factor V deficient plasma. Results: As previously reported by us, the animals exposed to hypoxic conditions demonstrated prothrombotic tendency as evident in significant shortening of clotting times (PT 84% of control, p 〈 .03) as compared to control animals. The exposed animals preinfused with PDI inhibitors demonstrated further reductions in clotting times. The preinfusion of Q-3R (0.25 to 1.5 mg/kg body wt) significantly shortened the Prothrombin times (71%, p 〈 .01) as compared to respective vehicle control. The hypercoagulative tendency triggered by Q-3R was also evident in drastically reduced activated clotting times in whole blood. Hypoxia induced the activation of PDI which was reflected by significantly elevated activity as compared to control animals. The dose dependent PDI inhibition in Q-3R preinfused animals was confirmed by the fallen activity of PDI in plasma from exposed animals. The calpain activity remained elevated in hypoxic animals (compared to controls) as observed previously. The oxidative damage as measured by MDA levels, was found to be much higher in exposed animals preinfused with Q-3R as compared to the vehicle group. Interestingly, the active factor V was observed to be significantly higher in case of PDI inhibition with largest activity increase in animals infused with highest dose of Q-3R. Conclusion:These results demonstrate the critical role of PDI in regulation of hypoxia induced prothrombotic state. The prevention of increase in activity of PDI in hypoxic animals by using specific PDI inhibitor accelerated the prothrombotic effect of hypoxia. These results appear to be in contrast with recently reported in vivo antithrombotic effect of PDI inhibition. However, as PDI upregulation is considered largely as a protective mechanism, this prothrombotic effect of PDI inhibition under hypoxia seems to be in part due to uncontrolled oxidative stress as shown by higher MDA levels. Also, as factor V activity was shot up by PDI inhibitors, this shows the activation of factor V to be under control of PDI, which can also be explained by role of disulfide linkages in activation of released factor V. Together, these results suggests a novel role of PDI, along with calpain, in regulating hypoxia induced prothrombotic phenotype. Disclosures No relevant conflicts of interest to declare.

Description: Background Treatment options for chronic lymphocytic leukemia (CLL) patients who received prior therapy are limited, especially for patients in high risk categories, such as del 17p; new therapies are urgently needed. In the Phase III RESONATE (Byrd, 2014), single agent ibrutinib, an oral, once-a-day, first-in-class covalent Bruton's tyrosine kinase inhibitor, was associated with improved progression-free survival (PFS, HR=0.215), overall survival (OS, HR=0.387) and overall response rate (ORR, 63% vs. 4%) compared with ofatumumab alone. Here we assessed the availability and comparability of clinical data for key therapies used in pre-treated CLL patients with ibrutinib and project long-term outcomes of ibrutinib for comparable studies using a simulation model. Methods A systematic literature review (SLR) identified relapsed or refractory CLL clinical trials on the following regimens: bendamustine ± rituximab (BR), fludarabine + cyclophosphamide + rituximab (FCR), ofatumumab, idelalisib + rituximab (IR), and rituximab. Patient populations and outcomes (ORR, PFS, and OS) were compared. A health state model was then developed to simulate long-term PFS and OS, comparing other therapies with ibrutinib. The model simulated treatment of a cohort of patients with pre-treated CLL until death or until disease progression, at which point they were simulated to receive subsequent treatment or best supportive care. Available Kaplan-Meier data were used to estimate longer-term projections of PFS and OS of comparable trials. Results The SLR identified few (n=36) relevant trials, only 4 of which were RCTs; comparison to ibrutinib through a common comparator was not possible. Pivotal trials was identified for each therapy based on the comparability to the RESONATE population, largest sample size and greatest amount of data reported. Selected trials (Table) showed significant heterogeneity between study populations, including variations in adverse cytogenetics (e.g. del 17p, del 11q), treatment line, and other characteristics (e.g. bulky disease) which have important ramifications on health outcomes. Three trials were excluded from simulation modeling: Robak (FCR) and LeBlond (BR) consisted of less heavily pre-treated patients than RESONATE; Furman et al. 2014 (rituximab and idelalisib+rituximab), enrolled a similar population to RESONATE aside from 17 p del, however, long-term safety and efficacy outcomes are limited due to the short follow-up in the idelalisib trial. Simulation over a 10 year horizon resulted in prolonged estimated mean OS for ibrutinib compared to ofatumumab (66 months versus 39 months). Despite the ibrutinib patients consisting of more heavily pre-treated, worse adverse cytogenetics and high risk features than those included in the BR and FCR studies, naïve comparison of long-term OS was projected to be 26 months and 8 months longer with ibrutinib respectively. Conclusion Comparisons of outcomes across trials are inherently limited by differences in populations, trial designs, and measurements. In this limited, naïve comparison of high risk CLL patients, ibrutinib is reasonably expected to provide incremental OS over BR and FCR in the pre-treated CLL setting, even when considering the RESONATE patient population included more heavily pre-treated patients and those with worse cytogenetics. The most rigorous comparison, using comparative data from a head-to-head trial, indicated that ibrutinib significantly prolonged PFS and OS compared to ofatumumab, which has established activity in hard-to-treat patients. Table. Patient Characteristics Patient Characteristics RESONATE RESONATE Fischer Badoux Furman Furman Robak FCR LeBlond BR Ibrutinib Ofatumumab BR FCR Rituximab + placebo Rituximab + idelalisib Sample Size 195 196 78 284 110 110 276 58 Median age (range) 67(30- 88) 67 (37- 88) 66.5 (42-86) 60 (NA) 71 (47-92) 71 (48- 90) 63 (35-83) 75 (49-87) Del 17p, (%) 32.30% 32.70% 19.20% 7% 28.20% 23.60% 7% 12% Median # of prior therapies (range) 3 (1-12) 2 (1-13) 2 (1-5)* 2 (1-10) 3 (1-9) 3 (1-12) 1 (1-1) 1 (1-2) % ≥3 prior therapies 52.80% 45.90% 43% 31% NA NA 0% 0% % Purine analog refractory 44.60% 44.90% 28.2%~ 19%~ NA NA NA NA Disclosures Peng: Janssen: Consultancy; Evidera: Employment. Pan:Janssen: Consultancy; Evidera: Employment. Sorensen:Evidera: Employment; Janssen: Consultancy. Dorman:Janssen: Consultancy; Evidera: Employment. Xu:Janssen: Consultancy; Evidera: Employment. Sallum:Evidera: Employment; Janssen: Consultancy. Gaudig:Janssen: Employment. Sengupta:Janssen: Employment. Wildgust:Janssen Global Services: Employment. Sun:Janssen: Employment.

Description: We report on a second generation of transgenic mice produced by crossing a transgenic mouse line expressing high levels of human alpha and beta S chains (alpha H beta S [beta MDD]) with a line expressing human alpha and beta S-Antilles (beta SAnt). We hypothesized that mice expressing both hemoglobins (Hbs) would have a more severe phenotype because the reduced oxygen affinity and solubility of the beta S- Antilles might enhance the rate and extent of polymer formation. We obtained mice that expressed both beta S and beta S-Antilles. The doubly transgenic mice that are heterozygous for deletion of mouse beta Major (beta MD) occurred with reduced frequency and those that are homozygous for deletion of mouse beta Major (beta MDD) occurred at a much reduced frequency and suffered early mortality. Human alpha was 58% of all alpha globin for all animals, whereas beta S and beta S- Antilles were 34% and 28% of all beta globins for beta MD mice and 42% and 36% for beta MDD mice. Hematocrit, Hb, and mean corpuscular Hb were normal for all transgenic mice, but reticulocyte levels were higher for the doubly transgenic mice versus alpha H beta S [beta MDD] mice older than 30 days (10.0% +/- 1.0% v 4.3% +/- 0.4%; P 〈 .001, mean +/- SE, n = 20 and n = 10, respectively) and control mice (3.9% +/- 0.4%). Reticulocytosis was more severe in mice less than 30 days old ( 〉〉 20% for alpha H beta S beta S-Ant[beta MDD] mice). The median mean corpuscular hemoglobin concentration of doubly transgenic mice was higher than that of alpha H beta S[beta MDD] mice with a variable number of very dense cells. Delay times for polymerization of Hb in red blood cells from alpha H beta S beta S-Ant[beta MDD] mice were shorter than those of alpha H beta S[beta MDD] mice, and there were fewer cells with delay times greater than 100 seconds. Urine-concentrating ability in control mice under ambient conditions is 2,846 +/- 294 mOsm and was reduced 30% to 1,958 +/- 240 mOsm, P 〈 4 x 10(-8) in all mice expressing both transgenes. We conclude that doubly transgenic mice have a more severe phenotype than either of the two parental lines. These mice may be suitable for validating therapeutic intervention in sickle cell disease.

Description: Abstract 1543 A growing body of evidence highlights the importance of microRNAs in regulating the expression of mediators of cell cycle progression. A theme emerging from these studies is that microRNAs participate in feedback or feed-forward circuits to provide bistability for key transition points in the cell cycle. We previously have shown that proper regulation of cyclin E activity is required for normal erythroid cell maturation in vivo, using cyclin ET74AüT393A knock-in mice, which have markedly dysregulated cyclin E due to its failure to interact with the Fbw7 ubiquitin ligase complex. We hypothesized that we could identify novel, microRNA-based molecular circuitry for maintaining appropriate levels of cyclin E activity by screening cyclin E knock-in erythroblasts for alterations in microRNA expression. We analyzed data we obtained from multiplex real-time PCR arrays comparing the expression of over 500 microRNAs in cyclin ET74A T393A knock-in versus wild-type erythroblasts (Ter119+/CD71+) and found down-regulated expression of a number of microRNAs targeting CDK inhibitors. We also identified down-regulated expression of potential microRNA regulators of Fbw7 expression. We found that overexpression of miR-223, in particular, significantly reduces Fbw7 mRNA levels, increases endogenous cyclin E protein and activity levels, and increases genomic instability. We next confirmed that miR-223 targets the Fbw7 3’ untranslated region. We then found that reduced miR-223 expression leads to increased Fbw7 expression and decreased cyclin E activity. Finally, we found that miR-223 expression in K562 cells is responsive to acute alterations in cyclin E regulation by the Fbw7 pathway and that dysregulated Fbw7 expression alters the erythroid differentiation capacity of these cells. Mir-223 plays an important role in myeloid and erythroid differentiation by regulating multiple substrates involved in these maturation programs. Here, we identify Fbw7 as a novel target of miR-223. Our data also indicate that miR-223 modulates Fbw7 expression as part of a homeostatic mechanism to regulate cyclin E activity and provide the first evidence that activity of the SCFFbw7 ubiquitin ligase can be controlled by the microRNA pathway. Disclosures: No relevant conflicts of interest to declare.