ALBERT

Description: We have previously designed and investigated novel allosteric effectors of hemoglobin (AEHs), as potential targeted treatment of sickle cell disease (SCD). In general, AEHs transiently covalently bind to hemoglobin (Hb), increase its affinity for O2, increasing the fraction of oxygenated sickle Hb (HbS), thus reducing HbS polymerization and countering red blood cell (RBC) sickling. In the current study, we designed a novel class of AEH molecules, incorporating a secondary mechanism of action (MOA), which is independent of Hb O2-affinity by interacting with the F-helix of deoxygenated HbS to directly destabilize its polymerization. Here, we report current results from our in-vitro and in-vivo studies with a representative AEH compound (PP-14). First, we assessed the anti-sickling properties in-vitro by incubating 0.5, 1, and 2 mM of PP-14 with whole blood suspensions from a subject with homozygous SCD (SS, hematocrit: 20%) under hypoxic conditions, with subsequent RBC sickling assessment by microscopy. Next, we subjected the samples to anoxia (100% N2 gas) to demonstrate the O2-affinity-independent antisickling mechanism. Subsequently, we tested residual samples for the degree of Hb modification (i.e., HbS-AEH adduct formation) and O2-affinity (p50) shifts. In a second experiment to further assess the secondary MOA, we subjected SS blood samples treated with various concentrations of PP-14 to hypoxia in the Hemox analyzer, which permitted us to obtain aliquot samples at defined pO2 values to establish pO2-dependent sickling. Additionally, we conducted in silico and in-vitro ADME studies to evaluate possible metabolic inhibition of a panel of CYP enzymes. Finally, we conducted a preliminary in-vivo PK/PD study in wild-type mice administered single doses of PP-14 via the oral (P.O.: 100-200 mg/kg) and intraperitoneal (I.P.: 75 mg/kg) routes. Serial blood samples were collected for up to 52 h after P.O., and up to 30 h after I.P. administration, and samples were assayed to quantify PP-14 concentrations. Residual blood samples were assayed for in-vivo Hb-AEH adduct formation, and the corresponding change in O2-affinity (Δp50, %). Our in-vitro studies demonstrated concentration-dependent inhibition of cell sickling of 25.5±11%, 44.4±3.8% and 90.8±1%, at 0.5, 1 and 2 mM of PP-14, respectively. HbS was modified correspondingly (38.9±9%, 55.7±4.9 %, and 92.4±9.8%), and was correlated linearly with the left-shift in OEC (Δp50 values of 11.3±5.1%, 29.0±13.2%, and 67.5±8.2%). Importantly, the antisickling effect was sustained under anoxic conditions (100% N2), strongly supporting the notion of a secondary, O2-affinity-independent MOA. Furthermore, we observed a dose-dependent delay in sickling, with initiation of sickling recorded at a pO2 level of 40 mmHg in absence of PP-14; and at 30 and 20 mmHg at 0.5, and 1 mM PP-14 concentrations, respectively. Complete inhibition of sickling was observed at 2 mM PP-14 through the lowest recorded pO2 level of 1.5 mmHg, a unique, effect not previously observed in any analogous AEH. In-vitro partitioning studies demonstrated that 〉90% of PP-14 partitioned into the RBC compartment when whole blood was incubated with 100-300 µM concentrations. Metabolic studies using pooled human liver microsomes (HLM) and isozyme-specific probe substrates suggested that up to 100 µM PP-14 did not inhibit CYP1A2, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP3A4 or CYP2B6. Wild-type mice after I.P. administration of PP-14 (75 mg/kg, n=5) showed peak concentrations in blood at 7 hrs (416.3±81.2 µM), with corresponding PD effects (Δp50 of 41.6±13.5%; modified Hb levels of 43.6±8.0%). Orally-treated mice had peak drug concentrations after 10-24 hrs, (~150 µM at 200 mg/kg, n= 2), with corresponding PD effects (Δp50 of 36.5±7.0%; modified Hb levels of 28.8±4.9%), which declined by 52 hrs. Overall, our data confirm that PP-14 is novel antisickling AEH with a secondary, O2-independent MOA in addition to the primary O2-dependent effect, as demonstrated by the inhibition of sickling under anoxic conditions. Additionally, PP-14 showed: excellent partitioning into the RBC compartment; acceptable in-silico ADME properties and in-vivo oral bioavailability; PD effects; and low liability for metabolic drug-drug interactions. Further studies to investigate formal detailed pharmacokinetic properties, and biological activity after single- or repeat-doses in a SCD mouse model are ongoing. Disclosures Safo: Sanofi: Consultancy, Research Funding; Virginia Commonwealth University: Patents & Royalties. Pagare:Virginia Commonwealth University: Patents & Royalties. Ghatge:Virginia Commonwealth University: Patents & Royalties. Rivella:Meira GTx, Ionis Pharmaceutical: Membership on an entity's Board of Directors or advisory committees; Disc medicine, Protagonist, LIPC, Meira GTx: Consultancy. Hines:Functional Fluidics: Equity Ownership. Liu:Functional Fluidics: Employment. Zhang:Virginia Commonwealth University: Patents & Royalties. Venitz:Virginia Commonwealth University: Patents & Royalties. Abdulmalik:The Children's Hospital of Philadelphia: Patents & Royalties: Provisional Patent.

Description: Megakaryopoiesis is a highly specialized cellular process which sustains platelet production. At the end of megakaryopoiesis, megakaryocyte (MKs) fragments into platelets via long and thin cytoplasmic extensions called proplatelets. Proplatelet formation (PPF) is associated essentially with cytoskeleton changes, including actin dynamics. The Rho/Rock pathway is a well characterized regulator of the actin reorganization. In the present study, we have tried to understand the precise role of the Rho/Rock pathway in PPF from human CD34+ derived MKs. Our results show that Rho is expressed in MKs and that its expression and activity remain stable during megakaryopoiesis. Overexpression of a RhoA dominant negatif (RhoA N19) in MKs leads to an increase in PPF. Conversely overexpression of a RhoA spontaneous active (RhoA V14) in MKs leads to a decrease in PPF. These results indicate that Rho activation could inhibit PPF in vitro. It is known that Rho/ROCK promotes actin cytoskeleton dynamics by regulating myosin light chain 2 (MLC2) phosphorylation. To demonstrate that Rho/Rock inhibits PPF through MLC2 phosphorylation, we added MLC kinase inhibitor (P18), Rho inhibitor (TatC3) and ROCK inhibitor (Y27362) in MKs culture just before PPF. Western blot analysis shows that MLC2 phosphorylation was inhibited by these 3 compounds, in contrast, PPF was significantly increased. Moreover, the platelet produced have an identical size and ultrastructure as control platelets and could be normally activated. These results suggest that Rho/ROCK could inhibit PPF through MLC phosphorylation during megakaryopoiesis.

Description: Dendritic cells (DCs) are highly specialized antigen-presenting cells that distribute widely in all organs. DCs initiate the primary immune response and activate naive T cells and B cells responsible for the acquired immunity. In this study, CCR7 mRNA was proved to be expressed in DCs and their precursors derived from murine bone marrow-derived hematopoietic progenitor cells (HPCs), whereas CCR1 mRNA was expressed in both CD11b−/dullCD11c+ and CD11b+hiCD11c+ DC precursors. CCR6 mRNA was not detected in any murine DC populations. In agreement with the chemokine receptor mRNA expression by each population in the DC differentiation pathway, SLC (also termed as MIP-3β), one of the ligands for CCR7, strongly and selectively chemoattracted both CD11b−/dullCD11c+ and CD11b+hiCD11c+ DC precursors (days 6 to 7) and more mature DCs (days 13 to 14). We have recently found that transforming growth factor-β1 (TGF-β1), a cytokine that is essential for the appearance of Langerhans cells in the skin, polarizes murine HPCs to generate Langerhans-like cells through monocyte/macrophage differentiation pathway. We observed here that TGF-β1 not only inhibited the expression of CCR7 in DCs and DC precursors derived from HPCs, but also inhibited the migration of these cells in response to SLC. This is the first report describing the chemokine and chemokine receptors responsible for murine DC migration and downregulation of DC migration by TGF-β1.

Description: Identifying tumor suppressor genes from intervals that are deleted in human hematologic malignancies such as 5q, 7q, 9q, and 20q has proven extremely challenging, and several laboratories have implicated haploinsufficiency as a likely mechanism. Chromosome engineering, which involves performing sequential rounds of gene targeting to insert loxP sites at flanking loci in mouse embryonic stem (ES) cells and using Cre recombination to delete the intervening sequences, was recently used to successfully interrogate the 1p36 interval in human solid tumors (Cell128(3):459–75, 2007). Monosomy 7 and deletion 7q [del(7q)] are among the most common cytogenetic alterations found in myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML). Cytogenetic analysis of patients who developed myeloid disorders with del(7q) uncovered a 2.5 Mb commonly deleted segment (CDS) within 7q22 (Blood88(6):1930–5, 1996), suggesting that this region plays an important role in leukemogenesis. To investigate the in vivo consequences of somatic loss of this interval, we generated a 5A3flox mouse model that harbor loxP sites flanking a ∼2 Mb interval on mouse chromosome 5A3 that is syntenic to the human 7q22 CDS. We intercrossed these mice with the interferon inducible Mx1-Cre transgenic strain, and injected these mice with polyinosinic-polycytidylic acid (pIpC) to delete the region in the hematopoietic compartment. The desired recombination is relatively inefficient; however, hematopoietic cells with loss of this region persist in the stem/progenitor compartment for over 1 year and are transplantable. We neither observed a block in differentiation nor clonal outgrowth of mutant hematopoietic cells, suggesting that additional mutations are necessary to initiate leukemia. We initiated AML in these mice by introducing additional genetic lesions using retroviral insertional mutagenesis, and we are characterizing these leukemias to study the effects of the 5A3 deletion on leukemogenesis and to clone cooperating genes. Chromosome engineering is a robust strategy for modeling leukemia-associated deletions in vivo, and for interrogating how loss of a specific interval alters hematopoietic growth. We are using the 5A3 strain to analyze candidate myeloid tumor suppressor genes from chromosome 7q and to uncover genes and pathways that cooperate in leukemogenesis.

Description: The combination of Imatinib and arsenic sulfide (As4S4) exerts more profound in vivo therapeutic effects on chronic myeloid leukemia (CML) at both organism and cellular levels, as evident for the first time in this paper by significantly prolonged lifespan in mouse leukemia model bearing BCR/ABL and induction of apoptosis of BCR/ABL expressing cells. To address mechanisms underlying this synergy, we performed systematic analysis of the dynamic change of proteome, phosphoproteome and transcriptome in K562 cells after As4S4 and/or Imatinib treatment with the support of principal component analysis (PCA) and self-organization maps (SOM). Moreover, protein biochemistry experiments were performed to confirm the important conclusions from multiomics study. The integrated information indicated that As4S4 promoted the unfolding protein reaction (UPR) and activity of ubiquitination pathway, which can be considered as a major biochemical basis of the pharmacological effects of this ancient medicine. In this context, As4S4 was shown to target BCR/ABL through ubiquitination of key lysine residues and led subsequently to its degradation by proteasome. Meanwhile, Imatinib inhibited the PI3K/AKT/mTOR pathway with synergism of As4S4 and arrested cell cycle. Combination of the two agents synergistically decreased activity and quantity of BCR/ABL and activated intrinsic and extrinsic apoptosis pathways. These complex multi-molecular target and multi-pathway modifications at protein level, together with those at transcriptional regulation level, ultimately resulted in the synergistic attenuation of BCR/ABL oncoprotein and the therapeutic effects on CML model.

Description: We investigate the frequency of the human leukocyte antigen DR15 (HLA-DR15) allele in patients with myelodysplastic syndromes (MDS). We used polymerase chain reaction-sequence-specific primers (PCR-SSP) to detect HLA-DR15 in the peripheral blood of patients with MDS(n = 76) and healthy controls (n = 227). The frequency of HLA-DR15 in MDS patients (40.8%) was significantly higher than in controls (13.7%; P 〈 0.01). The diagnoses of refractory anemia/refractory anemia with ring sideroblasts (RA/RARS) accounted for 77.4% (24/31) and 62.2% (28/45) of the DR15-positive and the DR15-negative patients, respectively (difference not statistically significant). Although no statistically significant difference was observed, some trends were observed: IPSS low-risk MDS (IPSS score, ≤1) accounted for 80.6% of the DR15-positive patients compared to 64.4% among the DR15-negative patients. However, the difference between the numbers of DR15-positive and DR15-negative patients with chromosomal abnormalities was not statistically significant. Nevertheless, poor risk chromosome abnormalities (according to IPSS), were present in only 1 DR15-positive patient, while such abnormalities were present in 8 DR15-negative patients. In addition, the proportions of DR15-positive and DR15-negative patients with more than 5% blasts in marrow were 19.4% and 31.1%, respectively. Peripheral blood pancytopenia occurred in 51.6% of DR15-positive, and in 40.0% of DR15-negative patients. Although the HLA-DR15 allele appeared to be present more frequently in patients less than 60 years of age, this association was not significant. The frequency of HLA-DR15 was significantly higher in patients with MDS than in healthy controls suggesting the possibility that HLA-DR15 is associated with an enhanced susceptibility to develop MDS. The fact that HLA-DR15 was predominantly noted in patients with RA/RARS and low IPSS scores, suggested that HLA-DR15 might be associated more with bone marrow failure and less with leukemic transformation. clinical/experimental characteristics in HLA-DR15 positive or negative MDS patients Cohort HLA-DR15 positive(n=31) HLA-DR15 negative(n=45) P value RA/RARS(case/%) 24/31(77.4) 28/45(62.2) 0.161 Low risk (IPSS≤1) (case/%) 25/31(80.6) 29/45(64.4) 0.126 Karyotype abnormal(case/%) 13/31(41.9) 18/45(40.0) 0.866 Poor chromosome(case/%) 1/31(3.2) 8/45(17.8) 0.117 Blast〉5%(case/%) 6/31(19.4) 14/45(31.1) 0.253 Pancytopenia(case/%) 16/31(51.6) 18/45(40.0) 0.317 Male patients(case/%) 17/31(54.8) 25/45(55.6) 0.951 age(

Description: It is now generally believed that proliferation of the neoplastic clone in chronic lymphocytic leukemia (CLL) takes place in lymphoid tissues where interactions involving the B-cell receptor (BCR) and other microenvironmental elements take place. Previous studies using in-vivo labelling with deuterated water have shown that recently proliferated emigrants from lymphoid tissues express low levels of the chemokine receptor CXCR4 and high levels of CD5 (CXCR4loCD5hi). It has been proposed that, following entry into the peripheral blood (PB), these cells become quiescent, re-express CXCR4 and downregulate CD5 allowing re-entry into tissues and further rounds of proliferation. In the present study we used in-vivo labelling with deuterated glucose (6,6-2H2-glucose, D2G) to investigate the proliferation and release of CLL cells into PB. In contrast to deuterated water, this technique allows pulse labelling of a distinct cohort of cells, which can then be tracked over time in-vivo. Labelling studies were performed in 10 patients with previously untreated, non-progressive CLL. Patients underwent 10 hours of labelling with oral 2DG, after which peripheral blood and lymph node compartments were serially sampled. DNA Deuterium enrichment was measured in the entire CLL population and in flow-sorted subsets defined by CXCR4/CD5 and surface IgM (sIgM) expression. Maximum release of labelled cells into PB occurred after a median of 14 days (4-56 days). The disappearance rate was very slow, with labelled cells detectable after 56 days in half of the subjects. In one case we were able to track labelled cells in both lymph node (LN) and PB and demonstrated an increase in the fraction of labelled cells in the LN between days 7 and 28, consistent with re-entry into this compartment. Subpopulations of PB CLL cells, defined by CXCR4 and CD5 expression were studied over time to investigate the dynamic nature of these molecules in circulating cells. As previously reported, maximum rapid incorporation of deuterium was observed in the CXCR4loCD5hi fraction, equivalent to 1.15 ± 0.04 %/d fractional synthesis at 7 days. Conversely, CXCR4hiCD5lo cells remained largely unlabelled throughout the 8-week study, reaching a maximum of only 0.01 ± 0.003 %/d, suggesting that they represent a non-proliferating population, not derived from the CXCR4loCD5hi subset. In contrast, CXCR4/CD5 intermediate cells exhibited delayed and intermediate labelling, peaking at 0.1 ± 0.02 %/d at 28 days. This sequential labelling pattern suggests that they derive from the CXCR4loCD5hi subset. Since both CXCR4 and CD5 expression are modulated by BCR signalling, we went on to sort cells according to sIgM expression and found maximum labelling in the sIgM high subset with little or no deuterium incorporation in the sIgM low fraction at any time. Again, the sIgM intermediate subset showed delayed labelling, suggesting that they are derived from sIgM-high cells. These observations provide further evidence for clonal heterogeneity in CLL and suggest the existence of distinct but interdependent subpopulations. Recently-divided cells are CXCR4loCD5hi, but appear to transition to an intermediate phenotype by down-regulation of CD5 and sIgM and upregulation of CXCR4 over the ensuing weeks, but do not appear to transition to CXCR4hiCD5lo cells over the time-course of the experiment. Our findings have clinical relevance, since these functionally distinct subsets might also differ in their responsiveness to therapeutic agents, such as drugs that block BCR signalling. Figure 1. Deuterium enrichment in CLL subsets over eight week study Figure 1. Deuterium enrichment in CLL subsets over eight week study Disclosures No relevant conflicts of interest to declare.

Description: Abstract 4561 Chronic myelomonocytic leukemia (CMML) typically has a dire prognosis with limited treatment options. We retrospectively reviewed the outcomes of 41 patients diagnosed with CMML (n= 35) or MDS/MPD overlap (n=6) who underwent allogeneic stem cell transplantation at three centers (University of Minnesota (n= 19), Johns Hopkins University (n=11), and the Cleveland Clinic (n=11)) between1990-2009. The majority of patients were male (59%) with a mean age of 51. At diagnosis nearly half of the patients had normal cytogenetics (n=20, 49%) with abnormalities of chromosome 7 the most commonly identified clonal finding (n=5, 12%). The majority had

Description: Whether chronic lymphocytic leukemia (CLL) represents latent or proliferating disease has been intensively debated. Whilst the dogma that CLL results from accumulation of dormant lymphocytes is supported by the unresponsiveness of leukemic cells to antigens and polyclonal activators, recent in vivo kinetic measurements show that B-lymphocytes do divide at significant rates in CLL. However, B cell kinetics were not compared between CLL patients and healthy controls so it was not possible to ascertain to what extent lymphocyte kinetics were aberrant in CLL. We compared proliferation rates of B- and T-lymphocytes in CLL patients and healthy controls, using a pulse-chase approach based on incorporation of deuterium from 6,6-2H2-glucose into DNA. We found dramatically reduced in vivo rates of CD3−CD19+ cell proliferation in CLL compared with controls (mean 0.47 versus 1.66 %/day respectively, P=0.001), equivalent to an extended half-life of circulating B-cells (147 days versus 42 days). Labeled (dividing) CD3−CD19+ cells had death rates similar to the healthy controls (2.29 versus 3.55 %/day, P=0.495). Despite such aberrant B-cells kinetics, T-cell proliferation was unaffected by CLL (1.77 versus 1.40 %/day, P=0.488). We conclude that, B-cell proliferation rates are reduced in leukemic patients compared to healthy subjects and that most circulating CD3−CD19+ cells are quiescent, long-lived cells.

Description: Sickle cell disease (SCD) continues to cause significant morbidity, mortality and healthcare disparities. Despite considerable progress in understanding the underlying pathophysiology and investigating various therapeutic strategies, novel pharmacologic approaches to ameliorate SCD continue to hold immense potential and promise, especially for patients in developing countries. Our group and others have recently renewed and refocused attention to candidate drugs that directly bind to hemoglobin (Hb) and increase oxygen (O2) affinity, preventing the fundamental pathophysiology of the disease, i.e., sickle Hb (Hb S) polymerization and red blood cell (RBC) sickling. While several candidate drugs have shown biological activity in-vitro, ex-vivo and in animal studies, their ultimate success in clinical studies was hampered by toxicity concerns and/or low oral bioavailability. Recent promising reports from a phase I/II study on 5-HMF renews optimism for this therapeutic approach. We reasoned that modifications of vanillin--a previously reported antisickling agent and food constituent without known toxicities--to enhance its efficacy, would represent a feasible approach in rationally developing clinically useful candidate drugs. Consequently, we designed and synthesized two classes of compounds: INN and TD series. The former are pyridyl derivatives of vanillin, rationalized to stereospecifically inhibit deoxy-Hb S polymer formation while increasing the fraction of the soluble oxy-Hb S in regions of low O2 tension. The TD compounds represent further modification of corresponding INN compounds (with a methoxyl group on the pyridine ring), rationalized to exhibit similar dual antisickling effects, but with enhanced direct polymer destabilization properties. We subjected a prototypical compound from each class (INN-270 and TD-7) to our battery of exploratory in-vitro assays, specifically: 1) rates of Hb S binding/modification, 2) corresponding change in O2 affinity, 3) direct inhibition of Hb S polymerization, and 4) inhibition of RBC sickling under hypoxia. We incubated 0.5, 1, or 2 mM of either INN-270 or TD-7 with RBCs from patients with homozygous SCD, under hypoxia (4% O2/96% N2 gas mixture) in a shaker-incubator at 37 ˚C for 3 h. Assays were conducted in at least three replicates utilizing different samples on different days. At the conclusion of each assay, aliquot samples (~ 10 μl each) were drawn into a fixing solution under hypoxia to preserve RBC morphology for analyses. Residual RBC suspensions were washed, hemolyzed, and subjected to: cation-exchange HPLC (to determine Hb modification); P50 analyses to establish change in O2 affinity; and temperature-dependent delay time studies to establish a delay in Hb S polymerization. Our results show that both compounds permeated RBC membranes without causing hemolysis, bound to and modified intracellular Hb at high levels in a dose dependent manner, increased O2 affinity significantly, and inhibited sickling of RBCs under hypoxia. TD-7 modified Hb S in a dose-dependent manner (to 92.3 ± 5.2 %, n=4 at 2 mM), shifted O2 equilibrium to the left (Δp50 = 45.6 ± 8.2 %, n=3 at 2 mM), and inhibited RBC sickling (by 95 -100 %, n=4). Preliminary delay time analyses also showed that at 2 mM, TD-7 increased the Hb S polymerization times from 18.1 ± 1.0 min to 24.5 ± 0.5 min. INN-270 showed a similar profile, however with a lower efficacy (at 2 mM) for Hb S modification (to ~ 75 %), Δp50 of 40.3 %, sickling inhibition by ~ 70 %, and increased delay times from 15.6 ± 0.5 min to 19.7 ± 1.0 min. We have elucidated the dual antisickling mechanism of action of INN-270 and TD-7 by X-ray crystallography. Two molecules of each compound bind to Hb via Schiff-base, and a series of hydrogen-bond/hydrophobic interactions that favor a high-O2-affinity Hb state. Importantly, the methoxyl group on the pyridine ring of TD-7 forms hydrogen-bond interactions with the surface-located αF-helix, resulting in a conformational change, possibly explaining the improved potency. Based on our results, both TD7 and INN 270 exhibited greater than a 40- and 3-fold superiority in efficacy compared to vanillin and 5-HMF, respectively. We conclude that our findings justify a prospective, structure-based approach to designing novel antisickling agents with enhanced potency. In-vitro/ex-vivo murine and human PK/PD studies are currently ongoing to help guide planned in-vivo PK/PD studies in mice. Disclosures Venitz: Consulted with AesRx LLC during phase I clinical studies of the antisickling compound, 5HMF for the treatment of sickle cell disease: Consultancy. Safo:Baxter and AesRx companies have licensed our patented antisickling compounds. Consulted with AesRx LLC during phase I clinical studies of the antisickling compound, 5HMF for the treatment of sickle cell disease: #7160910; #7119208 Patents & Royalties, Consultancy, Research Funding.