Mitchell Weiss, MD, PhD, Children’s Hospital of Philadelphia, Philadelphia, PA

Ralph Green, MD, PhD, University of California Davis Medical Center, Sacramento, CA

The study of autoimmune disease is greatly facilitated by focusing on disorders in which the associated autoantibodies are more than just markers of disease but directly contribute to the pathogenic process. By characterizing the autoimmune response on a molecular level, one can ask questions about the genetic restriction and clonality of the B-cell response which can increase our understanding of disease pathophysiology and lead to the development of better diagnostic and therapeutic agents. The acquired form of thrombotic thrombocytopenic purpura (TTP) is one such example of an autoimmune disease in which the pathology, i.e., platelet thrombosis, is linked to the development of autoantibodies. Such autoantibodies decrease the activity of ADAMTS13, a VWF-cleaving protease, resulting in the accumulation of "unusually" large VWF multimers that mediate platelet thrombosis. Antibody phage display has been used to clone the immune repertoires of four unrelated patients with the acquired form of TTP. A large number of patient monoclonal antibodies (mAbs) specific for ADAMTS13 were isolated by selection of phage libraries against recombinant ADAMTS13. Unique clones were identified by nucleotide sequencing, and their ability to interact with ADAMTS13 was characterized. To date, over 130 unique anti-ADAMTS13 antibodies have been analyzed and remarkably ~80 percent of antibody heavy chains show genetic restriction to the VH1-69 immunoglobulin (Ig) germline gene with the balance of heavy chains encoded by a variety of other genes from the VH1, VH3, and VH4 families. Interestingly, though all 130 antibodies bind to ADAMTS13 by ELISA, it appears that, with very few exceptions, it is only those antibodies encoded by VH1-69 that inhibit ADAMTS13 proteolytic activity, as measured by cleavage of the FRET-VWF73 substrate. Binding of human mAbs to ADAMTS13 was blocked by preincubation with murine plasma, suggesting crossreactivity with mouse ADAMTS13. Those mAbs that inhibited human ADAMTS13 activity in vitro inhibited murine ADAMTS13 in vivo after intravenous injection and subsequent measurement of murine plasma ADAMTS13 activity. Immunization of rabbits with human ADAMTS13-inhibiting mAbs generated anti-idiotypic antibodies that blocked antibody-mediated enzyme inhibition. In sum, these studies suggest that the humoral autoimmune response in TTP is genetically restricted, a property which, in theory, could be exploited for the treatment of TTP, i.e., the selective deletion of B-cells utilizing the VH1-69 heavy chain gene. Furthermore, crossreactivity of patient mAbs with murine ADAMTS13 may provide a mouse model of acquired TTP in which the role of autoantibodies in the presence and absence of environmental triggers may be studied. The cloning of anti-idiotypic mAbs from rabbit phage display libraries may serve as useful diagnostic tools to identify pathogenic autoantibodies in patient plasma and/or provide leads for the development of novel therapeutic approaches.

The accurate identification of an antiphospholipid antibody is a critical function of a clinical coagulation laboratory, since these autoantibodies impact treatment strategies for symptomatic patients as well as recommendations for asymptomatic individuals in whom antibodies are serendipitously found. Currently, two general methods are used to identify these antibodies: (1) coagulation assays to detect lupus anticoagulants, and (2) ELISA-based immunoassays, most commonly to detect anticardiolipin antibodies. Although new reagents and tests have been developed to improve the sensitivity and specificity of testing for lupus anticoagulants, the fundamental strategy remains unchanged: (1) prolongation of a phospholipid-dependent clotting assay; (2) lack of correction of the clotting time when patient plasma is mixed with normal plasma; and (3) confirmation of the phospholipid-dependent nature of the inhibitor. Detection of a lupus anticoagulant is affected by numerous variables, however, including anticoagulant therapy and sample preparation. Immunoassays for anticardiolipin antibodies can be used to screen large numbers of patient samples more easily, but are limited by inter-assay variability and lack of specificity. In 1990, three groups independently reported that most anticardiolipin antibodies actually bind to β₂-glycoprotein I, a phospholipid-binding plasma protein that modulates phagocytosis of apoptotic cells. Subsequently, several studies have shown that anti-β₂-glycoprotein I antibodies are more specific than anticardiolipin antibodies for the clinical manifestations frequently observed in these patients (e.g., thrombosis, pregnancy loss). In 2006, consensus guidelines for the diagnosis of the antiphospholipid syndrome (APS) were updated, and laboratory criteria include lupus anticoagulants, anticardiolipin IgG and IgM antibodies, and/or anti-β₂-glycoprotein I IgG and IgM antibodies, demonstrated on two (or more) occasions at least 12 weeks apart. Although many of these patients may have IgA anticardiolipin and/or anti-β₂-glycoprotein I antibodies, these are not considered diagnostic for APS. Many of these patients also have autoantibodies to prothrombin, although anti-prothrombin antibodies are not consistently associated with clinical manifestations and are not currently included in the laboratory diagnostic criteria for APS. More recently, diagnostic assays based on potential prothrombotic mechanisms of antiphospholipid antibodies, such as inhibition of the anticoagulant activity of annexin A5, have been described. As with anti-β₂-glycoprotein I antibodies, these assays appear to be more specific for antibodies detected in patients with clinical manifestations. We have recently used a gene expression strategy to identify unique gene expression signatures to distinguish patients with APS and venous thrombosis from patients with venous thromboembolism without evidence for antiphospholipid antibodies, as a strategy for identifying novel prothrombotic mechanisms that may form the basis of new clinical laboratory tests specific for these pathologic autoantibodies.

Michael P. Reilly, PhD, Thomas Jefferson University, Philadelphia, PA
Pathogenesis of Thrombosis in Heparin-Induced Thrombocytopenia

One of the most serious complications of heparin therapy is heparin-induced thrombocytopenia (HIT). HIT is the most frequently encountered drug-induced antibody-mediated thrombocytopenia. Extensive studies in vitro have shown that antibodies reactive with complexes of heparin and platelet factor 4 (PF4) lead to Fc receptor-mediated platelet activation. Patients who develop heparin/PF4 antibodies are at increased risk for life- and limb-threatening thrombosis. Laboratory methods to detect heparin/PF4 antibodies include enzyme immunoassays and functional assays. The former are readily available but lack specificity, while the latter have high specificity but are not readily available outside of specialized laboratories. However, the presence of antibodies is not always associated with thrombocytopenia or thrombosis. There is still little understanding of why a subset of patients with heparin/PF4 antibodies develops the disease. A critical gap in the field is the inability to distinguish thrombogenic heparin/PF4 antibodies from non-thrombogenic antibodies. In addition, little is understood about the host factors that contribute to thrombosis induced by immune complexes that contain the heparin/PF4 antibodies. Recent advances in understanding the pathology of HIT include the development of a transgenic mouse model of HIT and a novel method to clone authentic human heparin/PF4 antibodies. The FcγRIγA/hPF4 transgenic mouse model of HIT is an ideal tool for studying the complex and inter-related derangements of the inflammatory and coagulation systems that result in clinical thrombosis. An understanding of the host contribution to thrombosis in patients with HIT would enable clinicians to avoid giving heparin to the patients at highest risk. Little is known about the specific quantitative and qualitative characteristics of heparin/PF4 antibodies that determine which patients with HIT will develop thrombosis. Because heparin/PF4 antibodies isolated from patients with HIT are generally polyclonal and heterogeneous, it has been difficult to distinguish those antibodies with thrombogenic potential. We recently developed a novel means of cloning human antibodies from primary human B-cells from patients diagnosed with HIT. The combination of cloned antibodies and the mouse model will facilitate systematic testing in vivo to identify and characterize those antibodies with pathogenic properties. The ability to distinguish antibodies based on their thrombogenic potential would facilitate development of improved diagnostic tests and would be a first step towards creating therapeutics to prevent or alleviate thrombosis induced by HIT.

Co-Authors: Scott M. Dessain, MD, PhD, and Steven E. McKenzie, MD, PhD, Thomas Jefferson University, Philadelphia, PA

Nikhil C. Munshi, MD, Dana-Farber Cancer Institute, Boston, MA

Ghulam Mufti, DM, FRCP, King’s College, London, United Kingdom
Profiling Gene Expression

Myelodysplastic syndromes (MDS) are clonal disorders of hematopoiesis characterized by morphological dysplasia, ineffective hematopoiesis, and peripheral blood cytopenias, with progressive evolution to acute myeloid leukemia in approximately 25 percent of cases. The mechanism behind the pathogenesis and progression of MDS and is yet unknown. The advent of genome-wide microarrays has opened up an entire arena of molecular targets and potential genes that can be implicated in the pathophysiology of MDS. These enabling technologies can allow genome-wide molecular analysis of genomic DNA, mRNA, microRNA, and also proteins from dysplastic patient material. For a majority of MDS cases, diagnosis lies primarily from morphology and cytogenetic analysis. However, this is not always successful, as the dysplastic clone may be small and often exhibits poor growth due to increased apoptosis and incomplete metaphase separations. The advent of single nucleotide polymorphism (SNP) microarray has greatly increased the resolution of detectable genomic aberrations. Also, for the first time, it allows the detection of regions of DNA harboring uniparental disomy (UPD), which we have shown to be present in constitutional DNA and which may also harbor mutations and epigenetic dominance. The pathogenesis and disease progression can be explored by analyzing the transcriptome of the cells. It is crucial for the correct cell type to be used reflecting the dysplastic phenotype of MDS. In fact, expression analysis using DNA microarrays from various haemopoietic tem cells, progenitor cells, or lineage-committed mature cells has identified several genes and expression profiles unique to different MDS subtypes and, furthermore, highlighted specific pathways that may play a part in disease progression. The evolution of standard 3’ expression array into exon arrays extends the analysis to identify splice variants of genes that were previously unknown. The complexity of research technology has been exponentially increasing, and the analytical tools available to analyze and correctly annotate large amounts of data have also evolved. It is anticipated that the application of these novel technologies will help in both understanding the pathogenesis of heterogenous diseases, such as MDS, and also will allow us to dissect the mode of action for specific therapies, such as DNMT and HDAC inhibitors, and as agents, such as lenalinomide.

Jean-Pierre Issa, MD, University of Texas, M.D. Anderson Cancer Center, Houston, TX
Targeting the Molecular Lesions in MDS

There are few genetic lesions described in MDS to date, which has complicated the development of targeted therapies in that disease. However, many MDS patients present characteristic epigenetic changes specific to the neoplastic clone, and genes affected are likely to play a role in the etiology of advanced MDS and its progression to AML. Genes such as INK4B, CTNNA1 (which maps to the critical deletion on 5q31) and RIL (which also maps to 5q31) play important functions in cell cycle regulation, apoptosis, and signal transduction and are frequently silenced in MDS cells, raising the possibility of therapeutic targeting of silencing pathways to treat patients with MDS. Two silencing mechanisms have been successfully targeted clinically – DNA methylation and histone deacetylation. The DNA methylation inhibitors azacitidine and decitabine have substantial single-agent activity in MDS and are both FDA approved for this indication. The histone deacetylase inhibitor valproic acid also has demonstrated single agent activity in MDS. Combinations of hypomethylators and histone deacetylase inhibitors show in-vitro synergy and were promising enough in phase I/II studies to have entered randomized phase II/III studies. However, important questions followed these early successes. While targeted, these agents also have off-target activity (e.g., DNA damage) and their on-target activity is not necessarily specific for the genes altered in MDS. There are data in support of an epigenetic mechanism of action of the drugs including optimal activity at low doses, slow onset of responses and correlations between responses and epigenetic modulation (gene methylation and expression), but the issue is not fully resolved. A characteristic finding in using these agents is delayed clonal elimination and association of best responses with cytogenetic normalization. Thus, the effect of the therapy is not purely differentiation, and the mechanisms of clonal elimination remain under investigation. In addition, there are no current clinical or molecular predictors of response and while resistance to epigenetic therapy develops in nearly all patients, the mechanisms of resistance are unknown. Nevertheless, the high response rate to hypomethylating agents observed in some studies has raised substantial hope that epigenetic therapy will modify the natural history of the disease and thus provide a viable therapeutic option to those patients with MDS with the worse prognosis. Improving epigenetic therapy and identifying responding patients and mechanisms of resistance will all be key to developing a treatment program that could offer cure in this disease.

A. John Barrett, MD, FRCP, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD
The Role of Cytotoxic T Cells in MDS Pathology

It is now well established that there is an autoimmune component to the cytopenia of myelodysplastic syndromes (MDS). This assumption is based on clinical observation (notably the recovery from pancytopenia following immunosuppressive [IS] treatment) and laboratory studies showing suppression of marrow progenitor cell growth by clonally expanded CD8 T cells. Responders to antithymocyte globulin-based IS are mainly younger MDS patients, many of whom have trisomy-8. Such responders not only recover sustained and useful hematological function, but also have less progression to leukemia than similar MDS patients who have not received IS. Because trisomy-8 MDS is so frequently responsive to IS, we selected this subtype for detailed investigation. Most patients with trisomy-8 have marked oligoclonal CD8 T cell expansions revealed by spectratyping. Flow-sorted expanded T cell Vβ subsets show specific inhibition of trisomy-8 CFU-GM growth. After IS-induced hematological recovery, spectratypes tend to return to a normal distribution pattern, and the marrow morphology loses dysplastic features, despite the fact that trisomy-8 clones persist in the marrow and may even increase. Analysis of apoptotic pathways in trisomy-8 cells revealed that despite fas and caspase activation, trisomy-8 cells do not complete apoptosis probably because survivin and BCL-2 are upregulated. We hypothesize that this incomplete apoptosis is the basis for dysplastic morphology and confers a growth advantage for these clones over normal cells. Analysis of gene arrays in trisomy-8 showed increased expression of Wilms tumor-1 (WT-1) gene. CD8+ T cells from expanded Vβ families of HLA A*0201 individuals were found to contain up to 1.5 percent tetramer positive CD8+ T cells specific for the WT1₁₂₆ peptide. These findings raise the possibility of a unique immune driven mechanism for clonal dominance in trisomy-8 MDS through powerful T cell responses to the over-expressed tumor antigen WT-1. Whether similar mechanisms occur in patients with MDS, other than those with trisomy-8, remains to be determined.

Ralph Carmel, MD, New York Methodist Hospital, Brooklyn, NY

I. David Goldman, MD, Albert Einstein College of Medicine, Bronx, NY
Folate Transport Carriers, Receptor-Mediated Endocytosis, and the Molecular Basis of Hereditary Folate Malabsorption

The mechanisms of transport of folates into mammalian cells constitute an area of considerable interest focused largely on (1) the reduced folate carrier (RFC-SLC19A1), an anion exchanger, and (2) an endocytic process mediated by high affinity folate receptors, FRs. These transporters are ubiquitously expressed in murine and human tissues. However, the functional properties of these transporters have been known to be distinct from the properties of intestinal folate absorption and neither transporter is mutated in patients with hereditary folate malabsorption (HFM; OMIM 229050). This is a rare autosomal recessive disorder, first reported in 1961, caused by both impaired intestinal folate absorption and impaired folate transport into the central nervous system. This disorder results in severe anemia, immune deficiency, and often, but not always, neurological defects that present in early infancy. A Proton-Coupled Folate Transporter (PCFT; NP_542400) was recently identified, a member of the superfamily of solute carriers (SLC46A1) highly conserved in vertebrates, that mediates intestinal folate absorption. PCFT has a low pH optimum consistent with the acid pH at the microclimate of the duodenum and upper jejunum where folates are absorbed and this carrier is expressed. PCFT is also highly expressed in the proximal renal tubule, the sinusoidal membrane of hepatocytes, choroid plexus, cerebral capillary vessels, placenta and to a lesser extent, in many other tissues. To date, 10 individuals from nine families with the clinical diagnosis of HFM, have been shown to have mutations in PCFT that, with one exception, are all different. All mutations are homozygous except for one patient who had two different PCFT-mutated alleles traced back to a maternal and paternal grandparent. Mutations are at highly conserved amino acid residues distributed within exons one to four; the fifth exon is very small. While the basis for PCFT function in the low pH environment of the intestine is clear, and there can be substantial residual transport of some folates/antifolates even at pH 7.4, the widespread expression in many tissues suggests another function. One possibility is that PCFT is required for, or facilitates, FR function by providing a mechanism by which folates are exported from acidified vesicles into the cytoplasm during receptor-mediated endocytosis. It is of interest that prior to recent studies on PCFT, SLC46A1 was proposed to be (1) a heme carrier protein (HCP1) that mediates a pH-independent, low affinity process (hemin uptake K_m ~125 μM) and (2) the mechanism of iron absorption in the small intestine. However, it appears that the primary, if not only, function of SLC46A1 is folate transport. In four HFM patients in which data could be obtained, serum iron, iron-binding capacity, and ferritin were normal, and their anemia was completely corrected with high-dose oral, or lower-dose parenteral, 5-formyltetrahydrofolate (leucovorin).

Young-In Kim, MD, FRCP(C), University of Toronto, Toronto, Ontario, Canada
Biology and Epidemiology of Folate and Cancer

Folate is a water-soluble B vitamin that is present naturally in foods, whereas folic acid is the fully oxidized monoglutamyl form of this vitamin that is used commercially in supplements and in fortified foods. The sole biochemical function known for folate is mediating the transfer of one-carbon units involved in nucleotide synthesis and biological methylation reactions. In this role, folate may play an important role in cancer development and progression. Indeed, epidemiologic studies suggest that folate intake (dietary and supplemental) and blood folate levels are inversely associated with the risk of several malignancies including cancer of the colorectum, oropharynx, esophagus, stomach, pancreas, lungs, cervix, ovary, and breast and neuroblastoma and leukemia. The best epidemiologic evidence for the inverse association between folate status and cancer risk exists for colorectal cancer (CRC) and its precursor, adenoma. Collectively, epidemiologic studies suggest a 20-40 percent reduction in the risk of CRC and adenomas in individuals with the highest folate intake compared with those with lowest intake. However, animal studies suggest that folate possesses dual modulatory effects on colorectal carcinogenesis depending on the timing and dose of folate intervention. Folate deficiency has an inhibitory effect, whereas folate supplementation has a promoting effect on the progression of established colorectal neoplasms. In contrast, folate deficiency in normal colorectal mucosa appears to predispose it to neoplastic transformation, and modest levels of folic acid supplementation suppress, whereas supraphysiologic supplemental doses enhance, the development of cancer in normal colorectal mucosa. Although small human intervention trials have suggested potential beneficial effects of folic acid supplementation on biomarkers of CRC, more recent large intervention trials do not support these earlier observations. In the Aspirin-Folate Polyp Prevention Study, folic acid supplementation (1 mg/d for up to six years) increased the risk of developing advanced adenomas (OR=1.67; 95 percent CI, 1.00-2.80) and of developing multiple (>2) adenomas (OR=2.32; 95 percent CI, 1.23-4.35), compared with placebo in 1,021 subjects with previously resected adenomas. One explanation for this observation is that folic acid supplementation might have promoted the progression of already existing, undiagnosed preneoplastic lesions or adenomas missed on initial colonoscopy in these predisposed patients. Several potential mechanisms relating to the role of folate in one-carbon transfer reactions and consequent DNA synthesis and epigenetic regulations exist to support the differential effects of folate on the development and progression of CRC in the normal colorectum and in established colorectal preneoplastic and neoplastic foci. Population-based folic acid fortification, intended to prevent neural tube defects, and folic acid supplementation (consumed by up to 40 percent of the North American population), long presumed to be purely beneficial and believed to provide several health benefits, may have unintended deleterious influences on the development and progression of CRC. Given the incidence and mortality of CRC and the prevalence of adenomas (present in up to 50 percent of the population), whether or not folic acid fortification and supplementation promote the progression of preneoplastic foci to CRC is a legitimate, significant public health concern. Based on the lack of compelling supportive evidence and on the potential tumor-promoting effect, routine folic acid supplementation should not be recommended as a chemopreventive measure against CRC and other cancers. Furthermore, the potential tumor-promoting effect of the dramatically increased folate intake resulting from mandatory folic acid fortification in the United States and Canada should be carefully monitored.

Robert Clarke, MD, University of Oxford, Oxford, United Kingdom
Clinical Trials of Folate Supplementation: Where Do We Stand Now?

Nigel S. Key, MD, University of North Carolina, Chapel Hill, NC

Guillermina Girardi, PhD, Weill Medical College of Cornell University, New York, NY
Tissue Factor Enhances Inflammation in Antiphospholipid Antibody-Induced Pregnancy Loss

Fetal loss in patients with antiphospholipid antibodies (aPL) has been ascribed to thrombosis of placental vessels. However, we have shown that inflammation is an essential trigger of fetal injury in a mouse model of aPL-induced fetal loss. Complement component C5a and neutrophils (PMNs) are key mediators of aPL-induced fetal loss. In this study, we analyzed the role of the procoagulant tissue factor (TF) in this model. We have previously shown that increased TF expression depends on complement activation. By blocking TF in wild type mice with a monoclonal antibody (mAb) or studying low TF expressing mice, we found that TF is an essential mediator in this model. Treatment with mAb anti-TF 1H1 prevented aPL-induced inflammation and pregnancy losses [fetal resorption frequency (FRF percent): aPL vs aPL + 1H1: 39±7 vs 11±3, p<0.001]. Low TF mice were also protected from aPL-induced inflammation and fetal death [(FRF percent): aPL (wild type mice): 41±3 versus aPL (low TF mice): 10±4 vs, p<0.005]. Low TF females that mated with wild type males were protected from aPL-induced fetal loss to a similar extent as low TF females who mated with low TF males, indicating that maternal TF mediated aPL-induced fetal injury. Given that PMNs are critical mediators of fetal death in this model, we tested the hypothesis that aPL generated-C5a induces TF expression on PMNs in vivo. We used FACS analysis to measure TF expression on peripheral blood PMNs from aPL-treated C5aR^+/+ and C5aR^-/- . PMNs from aPL-treated C5aR^+/+ mice were 39±9 percent positive for TF compared to 9±3 percent of the PMNs in C5aR^-/- mice and 5±2 percent of PMNs from untreated mice. To investigate the role of maternal myeloid cells TF in aPL-induced pregnancy loss, we performed studies in TF ^{floxed/floxed}/LysM-Cre mice, in which the TF gene is selectively deleted in myeloid cells. Lack of TF expression on PMNs was confirmed by FACs. aPL did not increase FRF in TF ^{floxed/floxed}/LysM-Cre mice (FRF percent:12±3). In contrast, a four-fold increase in FRF was observed in TF ^{floxed/floxed} mice treated with aPL (FRF percent:39±6). The lack of TF expression on neutrophils completely rescued embryos from aPL-induced injury and diminished neutrophil infiltration in deciduas. Moreover, aPL-treated TF ^{floxed/floxed}/LysM-Cre mice showed diminished reactive-oxygen production in neutrophils and less free-radical-mediated lipid peroxidation in decidual tissue when compared to aPL-treated TF ^{floxed/floxed} mice, suggesting that TF on neutrophils contributes to respiratory burst potentiating neutrophil-mediated trophoblast injury and fetal loss. Taken together, our data indicate that expression of TF on neutrophils is a crucial mediator of aPL-induced pregnancy loss and that lack of TF expression on neutrophils prevents inflammation and averts the fetal resorptions characteristic of aPL pregnancies. The identification of neutrophil TF as an important effector in aPL-induced inflammation may allow the development of new therapies to abrogate the inflammatory loop caused by complement and TF and improve pregnancy outcomes in patients with aPL.

John H. Griffin, PhD, Scripps Research Institute, La Jolla, CA
Cytoprotective Activities of Activated Protien C

Plasma Protein C is known for its mild deficiency linked to venous thrombosis risk and severe deficiency linked to neonatal purpura fulminans. Activated protein C (APC) anticoagulant activity involves proteolytic inactivation of factors Va and VIIIa, and venous thrombosis risk linked to APC resistance is due to factor V Leiden. The clinical success of recombinant APC in reducing mortality in severe sepsis patients (PROWESS trial) gave impetus to new directions for basic and pre-clinical research on APC. Remarkable insights from recent in vitro and in vivo studies are centered on the direct cytoprotective effects of APC that include beneficial alterations in gene expression profiles, anti-inflammatory actions, anti-apoptotic activities, and stabilization of endothelial barriers. Independent of anticoagulant effects, these cytoprotective effects generally require the two receptors, endothelial cell protein C receptor (EPCR) and protease-activated receptor-1 (PAR1). Because of its pleiotropic activities, APC has potential roles in the treatment of complex disorders, including inter alii sepsis and ischemic stroke. Recent work reveals the relative importance of APC's various activities for efficacy in murine sepsis and ischemic stroke models. When genetically altered mice deficient in APC receptors and protein engineering of murine APC were employed in murine model studies, mortality reduction in LPS-induced endotoxemia required the enzymatic active site of APC, EPCR and PAR1, highlighting a key role for APC’s cytoprotective actions. Consistent with this, an APC variant engineered to have normal cell-signaling but less than 8 percent anticoagulant activity (5A-APC) was similar to wild-type APC in reducing mortality after LPS challenge or peritonitis induced by gram-positive or gram-negative bacteria or by colon damage. Murine ischemic stroke and NMDA-excitotoxic injury model studies showed that APC's neuroprotective effects require EPCR and PAR1 and are, at least in part, independent of APC's anticoagulant activity. Thus, APC’s efficacy for mortality reduction in sepsis and for neuroprotective actions appears to be based predominantly on EPCR-dependent and PAR1-dependent cell signaling. One may speculate that APC variants with normal cell-signaling but reduced anticoagulant activities might prove beneficial while having reduced bleeding risk in treating sepsis and stroke. In summary, recent data underscore the cytoprotective actions of APC as key for its therapeutic efficacy.

References:
Mosnier LO, Zlokovic BV, Griffin JH. The cytoprotective protein C pathway. Blood. 2007;109:3161-72.

Kerschen EJ, Fernandez JA, Cooley BC, et al. Mechanisms for endotoxemia and sepsis mortality reduction by non-anticoagulant activated protein C. J Exper Med. 2007;in press.

Cheng T, Petraglia AL, Li Z, et al. Activated protein C inhibits tissue plasminogen activator-induced brain hemorrhage. Nature Med. 2006;22:1278-85.

Guy Zimmerman, MD, Eccles Institute of Human Genetics, University of Utah, Salt Lake City, UT
Platelet-Leukocyte Interactions in Inflammation and Thrombosis

Activated human platelets adhere to and interact with myeloid leukocytes in vivo and in vitro. In vivo, heterotypic platelet-leukocyte aggregates form in the blood of subjects with sepsis, diabetes, acute coronary syndromes, and in other conditions of vascular injury, and are sensitive markers of platelet activation in the blood. In addition, platelet-leukocyte aggregates circulate in murine models of disease. Platelets also interact with monocytes and polymorphonuclear leukocytes in thrombi and at sites of inflammation. In vitro, molecular analysis demonstrates that adhesive interactions between platelets and myeloid leukocytes are mediated by specific binding of P-selectin displayed by the activated platelets to P-selectin glycoprotein 1 (PSGL-1) on the leukocyte plasma membranes. Stable cell-cell adhesion and functional responses result. For example, platelet-monocyte interaction via P-selectin/PSGL-1 is reported to induce Tissue Factor expression and synthesis of procoagulant cytokines. This illustrates the intimate links between hemostasis and inflammation, and diverse repertoires of platelets and leukocytes as hemostatic and inflammatory effector cells. In vitro studies demonstrate that engagement of PSGL-1 by P-selectin transmits outside-in signals to transcriptional and post-transcriptional pathways of gene expression in monocytes, alone or in concert with other adhesion pathways and/or with signaling by paracrine factors released by the activated platelets. Autocrine signaling by monocyte factors may also modulate specific checkpoints. Using models of human platelet interactions with monocytes, we have identified patterns of newly-expressed inflammatory and prothrombotic proteins that are synthesized as a result of these cellular dialogues. We have also defined previously-unrecognized transcriptional and post-transcriptional regulatory mechanisms in both the platelet and the monocyte that are involved in these synthetic and functional responses. Thus, platelet-leukocyte interactions are clinically relevant events that also provide robust experimental systems for elucidating mechanisms of gene expression in specialized human cells. The findings to date indicate that additional expressed genes and regulatory pathways remain to be discovered, and also suggest that these pathways are therapeutic targets in inflammatory and thrombotic syndromes.

Co-Authors: Kristi Bemis-Standoli, PhD, Dan Dixon, PhD, South Carolina Cancer Center, University of South Carolina, Columbia, SC; Melvin Denis, PhD, Jason Foulkes, PhD, Estelle Harris, MD, Larry Kraiss, MD, Tracey Mahoney, PhD, Mark Martinez, MD, Noemi Michetti, Hansjörg Schwertz, MD, Neal Tolley, MS, Andrew Weyrich, PhD, Eccles Institute of Human Genetics, University of Utah, Salt Lake City, UT; Christopher Hanrahan, MD, PhD, Kathryn Morton, MD, University of Utah, Salt Lake City, UT; Stephan Lindemann, MD, Medizinische Klinik III, Eberhard Karls Universitat Tubingen, Tubingen, Germany; Thomas McIntyre, PhD, Lerner Research Institute, Cleveland, OH; and Stephen Prescott, MD, Oklahoma Medical Research Foundation, Oklahoma City, OK

Mario Cazzola, MD, University of Pavia Medical School, Pavia, Italy

Carole Beaumont, PhD, INSERM U773, Centre de Recherche Biomédicale Bichat Beaujon, Paris, France
Erythrophagocytosis and Recycling of Heme Iron in Normal and Pathological Conditions

Iron homeostasis relies mostly on the efficient and tightly controlled recycling of iron by tissue macrophages following phagocytosis of senescent red blood cells. This recycling of heme iron provides most of the iron required daily for bone marrow erythropoiesis. Throughout their lifespan, circulating erythrocytes will accumulate biochemical changes at their surface, such as peroxydation of membrane-bound lipoproteins, loss of syalic acid residues, and formation of senescence neoantigens, or will undergo eryptosis, a particular programmed cell death characteristic of red blood cells. These modifications will allow tissue macrophages from bone marrow, spleen, and liver to identify the red blood cells to be eliminated, through interactions with specific receptors. After this initial recognition step, the red blood cell is internalized by phagocytosis, and the phagosome undergoes a series of fusion with intracellular vesicles and with the endoplasmic reticulum to acquire the machinery necessary for the degradation of red cell constituents. The heme molecule is catabolized by an enzymatic complex anchored in the endoplasmic reticulum membrane and comprising an NADPH-cytochrome c reductase, heme oxygenase 1, and biliverdin reductase. Iron released from heme catabolism is either recycled back to the plasma through ferroportin, a membrane-bound Fe (II) export molecule, or retained within the ferritin molecules, to be released at later stages. Erythrophagocytosis induces changes in macrophage gene expression, including heme oxygenase 1, ferroportin, and ferritin, by several mechanisms. Heme is a potent transcriptional activator of heme oxygenase 1 gene and probably also of the ferroportin gene, whereas iron released from heme catabolism regulates ferroportin and ferritin mRNA translation, through the IRE/IRP system. However, the actual amount of iron exported from macrophages to the plasma is directly controlled by hepcidin through its interaction with ferroportin. Hepcidin is a peptide hormone acting as a negative regulator of iron homeostasis and allowing a fine-tuning of the amount of iron recycled by macrophages to the iron demand of bone marrow erythropoiesis. Modifications in erythrophagocytosis contribute to the pathogenesis of several disorders. Increased eryptosis of erythrocytes has been described in iron deficiency in sickle cell disease or in patients with non-alcoholic steatohepatitis, contributing to reduced lifespan of red blood cells, severity of the anemia, and abnormal liver iron deposits. Activation of macrophages by cytokines and increased erythrophagocytosis is a hallmark of the anemia of chronic diseases and of the hemophagocytic syndrome. Therefore, erythrophagocytosis and recycling of heme iron appears as a central process in iron homeostasis.

Elizabeta Nemeth, PhD, University of California – Los Angeles, Los Angeles, CA
Ferroportin Function and Ferroportin Disease

Most if not all iron enters plasma through ferroportin, the only known cellular iron exporter in vertebrates. Ferroportin is a transmembrane protein expressed in all tissues that handle major iron flows: macrophages, which recycle old red blood cells; hepatocytes, which store iron; duodenal enterocytes, which absorb iron; and placental trophoblasts, which transfer iron from mother to fetus during fetal development. Details of ferroportin structure and function are still unresolved, including its membrane topology and the mechanism of iron export. The regulation of ferroportin levels is somewhat better understood: ferroportin is mainly posttranslationally regulated by the hepatic hormone hepcidin. Hepcidin binds to ferroportin and causes its internalization and degradation. Recent studies indicate that the process of internalization is dependent on the phosphorylation of ferroportin, and that subsequent ubiquitination targets ferroportin for degradation in lysosomes. Ultimately, the loss of ferroportin from the cell surface results in decreased iron flows into plasma. The hepcidin-ferroportin interaction is critical for normal iron homeostasis and underlies the pathogenesis of iron disorders including anemia of inflammation, iron-loading anemias, and hereditary hemochromatosis. Ferroportin also appears to be directly regulated by iron and inflammation on transcriptional and possibly posttranscriptional level, but the relative biological significance of these processes remains to be determined. Complete loss of ferroportin was shown to cause embryonic lethality in zebrafish and mice. Heterozygous missense mutations in the ferroportin gene cause an autosomal-dominant form of iron overload called type IV hemochromatosis or "ferroportin disease." However, "ferroportin disease" has a varied clinical presentation due to different ferroportin mutations with distinct consequences for iron transport. One group of mutations causes a defect in trafficking where the mutant protein is retained inside the cell resulting in a loss of iron-exporting function. Patients carrying these mutations accumulate iron in macrophages, which leads to high ferritin levels, low-to-normal transferrin saturation, and possibly borderline anemia, particularly when phlebotomized. Another group of mutations prevents hepcidin-mediated internalization and degradation of ferroportin resulting in a "gain-of-function." The most severe of these are the mutations affecting the C326 residue which interfere with hepcidin binding. As a result, patients develop a phenotype similar to classical hemochromatosis caused by hepcidin deficiency, with increased duodenal iron absorption, high transferrin saturation, and iron deposition in hepatic parenchyma and other tissues. The mechanism of dominant inheritance of ferroportin disease appears to be due to ferroportin functioning as a multimer. Disease-causing mutants may act as dominant negatives and interfere with the trafficking or with hepcidin-mediated degradation of normal ferroportin. The nomenclature of ferroportin diseases should be revised to reflect the clinical differences between ferroportin mutations that cause parenchymal versus macrophage iron loading.

Clara Camaschella, MD, Universita Vita-Salute San Raffaele, Milano, Italy
A New Model of Anemia Due to Defective Mitochondrial Iron Metabolism

Rare inherited anemias strengthen the relationship between mitochondrial and cytosolic iron and support a link between the two pathways of mitochondrial iron utilization by erythroid cells, heme, and iron sulfur (Fe/S) clusters synthesis. X-linked sideroblastic anemia is due to mutations of aminolevulinic acid-synthase 2 (ALAS2), which catalyzes the first step of heme synthesis in erythroblasts and is postranscriptionally regulated by Iron-Regulatory-Proteins (IRP1 and 2) through its 5’UTR Iron Responsive Element (IRE). X-linked sideroblastic anemia with ataxia is due to defective ABCB7, a mitochondrial exporter of Fe/S clusters. The mitochondrial enzyme glutaredoxin-5 (GLRX5) participates to the biogenesis of Fe/S clusters in yeast and zebrafish. Its deletion in the zebrafish mutant shiraz is lethal because of severe anemia, due to the absence of Fe/S clusters-dependent aconitase, upregulation of IRP1, and inhibition of IRE-ALAS2. Human GLRX5 rescues the shiraz phenotype, suggesting an evolutionary conserved function. We have identified the human counterpart of shiraz in a middle-aged male patient with longstanding microcytic-hypochromic anemia, low percentage of ringed sideroblasts, and severe clinical complications of iron overload.¹ Over time, he required an increasingly intensive blood transfusion regimen; anemia was remarkably improved, up to transfusion-independency, after iron chelation. Patient GLRX5 had a homozygous (c.294 A->G) splice mutation, which results in severe RNA reduction. Studies of patient peripheral blood mononuclear cells (PBMC) and a lymphoblastoid cell line (LCL) showed low cytosolic aconitase, high IRP1 bindingactivity, low ferritin, and high transferrin receptor. Mitochondrial Fe/S cluster enzymes activities were reduced in PBMC, but normal in LCL. GLRX5 activity appears sufficient in nonerythroid cells with non-IRE ALAS1, but inhibition of IRE-ALAS2 in erythroblasts causes heme reduction and anemia, as in shiraz. Moreover, it originates a vicious circle between low heme (anemia) and iron overload. We reasoned that the IRP1 defect cannot explain the Hb variations and hypothesized a contribution of IRP2, whose proteasome degradation is iron- and heme-mediated. IRP2, expected to be upregulated in GLRX5 deficiency because of low heme, contributes to ferritin and ALAS2 repression and to increased mitochondrial iron. Accordingly, iron chelation, redistributing iron to the cytosol, might relief IRP2 excess, improving heme synthesis and anemia. This hypothesis is in agreement with the essential role of IRP2 in erythroid iron uptake, as shown by Irp2-deficient mice. Perturbation of Fe/S clusters synthesis in humans leads both to anemia and to iron overload, indicating that iron homeostasis is regulated by mitochondrial iron utilization. The proposed new model of anemia, worsened by iron overload and improved by iron chelation, might account for other congenital/acquired sideroblastic anemias ameliorated by iron depletion.

1. Camaschella C, Campanella A, De Falco L, et al. The human counterpart of zebrafish shiraz shows sideroblastic-like microcytic anemia and iron overload. Blood. 2007;110:1353-8.

Co-Authors: Alessandro Campanella, Sonia Levi, PhD, and Laura Silvestri, Vita-Salute University - IRCCS San Raffaele Milan, Italy; Achille Iolascon, MD, Federico II University, Naples, Italy

Systems Biology

Riccardo Dalla-Favera, MD, Institute for Cancer Genetics, Columbia University, New York, NY

Kimberly Stegmaier, MD, Dana-Farber Cancer Institute and Children’s Hospital Boston, Boston, MA, and Broad Institute of Harvard University and Massachusetts Institute of Technology, Cambridge, MA
Signature-Based Approaches to Leukemia Pathogenesis and Therapy Discovery

Despite progress in understanding the pathogenesis of hematological malignancies, translation of scientific knowledge to clinical benefits remains slow. The genomic revolution has enabled a more granular approach to disease characterization, yet therapy development still lags behind. Traditional approaches to small molecule discovery have largely focused on simple phenotype-based (i.e., cell-death assays) or target-based screens. Each of these has played an important role in the drug discovery process, but each also has its limitations. As such, a host of challenges continue to impede compound discovery efforts: (1) Current methods of small molecule library screening have been limited; (2) Cell-free biochemical assays do not recapitulate the complexity within a cell; (3) Many potential targets have been considered pharmacologically intractable; and (4) Often the target of disease pathogenesis is not known. A more systems-based approach to small molecule library screening has the potential to overcome these challenges. With the sequencing of the human genome and the advent of genome-wide expression profiling, alternative approaches to chemical biology become feasible. Several new tactics have been explored, each using gene expression signatures to facilitate compound discovery. Our initial efforts focused on the use of multi-gene signatures as proxies for highly complex cellular networks in a small molecule library screen. In Gene Expression-based High-throughput Screening (GE-HTS), gene expression signatures of the biological states of interest (i.e., "state A" versus "state B") are first defined using genome-wide expression profiling. Next, ligation-mediated amplification with fluorescent-bead based detection of amplicons is used for the high-throughput, low-cost measurement of the signature. Then, a small molecule library is screened for compounds that induce a change from the "state A" to the "state B" signature. Unlike traditional phenotype-based screens, GE-HTS does not require development of specialized assays. The signature definition, amplification, and detection are generic. Furthermore, a priori knowledge of a target is not needed because the signature serves as a surrogate for the biological state in question. Our first proof of principle experiments successfully applied GE-HTS to the identification of compounds that induce differentiation in acute myeloid leukemia. A second signature-based approach utilized a reference collection of gene expression profiles from human cells treated with bioactive small molecules. Here, the goal was to create an in silico tool (the Connectivity Map) for discovering the relationships among diseases, physiological processes, and small molecule interventions. A pilot study demonstrated the feasibility of this approach with the expression profiling of 164 distinct small molecule perturbagens. With the genomic revolution, it is feasible to consider small molecule library screening in the absence of detailed molecular understanding of a disease. Signature-based approaches should enable systematic exploration of diseases and biological processes not previously studied and the identification of new therapeutic leads.

Co-Authors: Todd R. Golub, MD, Broad Institute of Harvard University and Massachusetts Institute of Technology, Cambridge, MA, and Dana-Farber Cancer Institute, Boston, MA, and Justin Lamb, PhD, and Kenneth N. Ross, PhD, Broad Institute of Harvard University and Massachusetts Institute of Technology, Cambridge, MA

Andrea Califano, Dr, Columbia University, New York, NY
Dissection of Dysregulated Cellular Networks for the Identification of Causal Lesions in Lymphoid Malignancies

The identification of genes that are causally related to the presentation of a specific malignant phenotype is still an open problem in cancer research. Systems Biology has started to produce a variety of molecular interaction maps – including transcriptional, complex-formation, metabolic, and signaling interaction – that should start to provide a rational basis for the systematic identification of these lesions. Unfortunately, these maps are still mostly limited to prokaryotic and lower eukaryotic organisms, and, even in that context, a truly integrated map remains quite elusive. Our lab has pioneered the development of information theoretic methods to predict both transcriptional (ARACNe) and post-translational (MINDy) interactions in human B cells. Predictions from these methods have been biochemically validated in vivo and shown to have low false-positive rates, comparable to those of more traditional experimental methods. By combining these methods with a variety of high-throughput experimental evidence and literature datamining tools – using a standard Bayesian evidence integration approach – we have produced the first comprehensive draft of a human B lymphocyte cellular network. The resulting B Cell Interactome (BCI) includes regulatory interactions (both for transcriptional and for signaling networks) as well as protein-protein interactions in complexes for a total of almost 200,000 interactions. We will discuss how such a draft can be used to produce maps of the dysregulated subnetworks in specific pathologic or physiologic phenotypes and how the latter can help to identify causal lesions in several well-studied B cell malignancies as well as key effectors of normal physiology. Finally, we will discuss the use of the BCI in the identification of molecular targets of specific drugs as well as in the selection of druggable targets to activate or inhibit specific genetic programs in B cells.

Louis M. Staudt, MD, PhD, National Cancer Institute, National Institutes of Health, Bethesda, MD
Achilles Heel RNA Interference Screens for Molecular Targets in Cancer

Inder Verma, PhD, The Salk Institute for Biological Sciences, San Diego, CA
NF-κB Signaling in Lymphoid Neoplasia

NFκB signaling plays a pivotal role in several cellular and developmental processes in metazoans. Deregulation of this pathway is suspected in and also causally linked to the initiation and progression of many human pathologies. Since its discovery in the B cells and the relatively greater understanding of its role in the cells of the immune system, uncontrolled NFκB activity has most often been associated with inflammatory disorders. NFκB is a family of transcription factors found associated with IκB family of inhibitory proteins and are predominantly localized in the cytoplasm. The common denominator for NFκB activation is the removal of IκB proteins from the DNA binding subunits of NFκB. The fact that NFκB is activated by over 200 stimuli, and it, in turn, activates an equally large subset of target genes in different cell types poses a major challenges in designing pathway specific inhibitors of NFκB. Knowledge of all relevant modifications of NFκB pathway members is imperative to comprehend how specificity is generated in such a promiscuous pathway and this would be critical in designing inhibitors that do not have pleiotropic effects. Inhibition of NFκB in various cellular compartments can be achieved by a) inhibiting the receptors and the adaptors on the membrane, b) the upstream kinases and IKKs, in the cytoplasm, and c) NFκB DNA binding and transactivation in the nucleus. Although the role of NFκB in regulating molecules involved in inflammation might be the primary unifying mechanism underlying most of the diseases, other activities of NFκB that aid in the development of diseases have now been uncovered. One prominent activity attributed to NFκB that might contribute towards its role in some human disorders is its ability to protect cells from apoptosis in response to a diverse set of physiological stimuli. Indeed the relevance of NFκB in mitigating apoptosis is now becoming evident in the evolution of several human malignancies. These malignancies include those of hematological origin, helicobacter pylori-associated carcinogenesis, and cancer of the breast, colon, liver, and cervix. Similarly, NFκB has been documented to play important role in subverting p53 induced cell death in lymphocytes. As evidence mounts that NFκB-mediated regulation of p53 is important in regulating apoptosis resistance and transformation, repression of NFκB by tumor suppressor genes, including p53, is also emerging as a potential mechanism of inhibiting apoptosis resistance. The knowledge of how specific modifications regulate NFκB activity in response to distinct stimuli would undoubtedly propel our efforts towards designing more specific inhibitors of NFκB.

Zhijian Chen, PhD, University of Texas Southwestern Medical Center at Dallas, Dallas, TX
Ubiquitin Signaling in the NF-κB Pathway

Ubiquitination plays an important role in NF-κB activation by targeting the inhibitor of NF-κB (IκB) for degradation by the proteasome. Ubiquitination also plays another role in the NF-κB pathway by activating several protein kinases, including TAK1 and IKK, through a proteolysis-independent mechanism. We found that TRAF proteins, including TRAF2 and TRAF6, which are crucial for NF-κB activation in different pathways, are RING domain ubiquitin ligases (E3s). These E3s function in conjunction with the ubiquitin-conjugating enzyme (E2) complex, consisting of Ubc13 and Uev1A, to catalyze K63-linked polyubiquitination of target proteins, including RIP1 and TRAF proteins themselves. The K63 polyubiquitin chains function as a scaffold to recruit the TAK1 and IKK complexes through binding to TAB2 and NEMO, the essential regulatory subunits of these kinase complexes, respectively. The assembly of TAK1 and IKK complexes on the polyubiquitin chains allows TAK1 to phosphorylate and activate IKK. This ubiquitin-dependent mechanism of protein kinase activation is key to NF-κB regulation in different signaling pathways, including those emanating from the antigen receptors in T and B lymphocytes. Dysregulation of ubiquitin signaling in the NF-κB pathways has been linked to some forms of lymphoid cancers, such as MALT lymphoma. Recent progress in understanding the mechanisms of ubiquitin-mediated activation of protein kinases will be presented.

Peter Leif Bergsagel, MD, Mayo Clinic, Scottsdale, AZ
The Emerging Role of Deregulated NF-κB Signaling in the Pathogenesis of Multiple Myeloma

Primary genetic abnormalities in multiple myeloma (MM) include recurrent immunoglobulin gene translocations involving 11q13, 6p21, 4p16, 16q23, and 20q11, and hyperdiploidy associated with trisomies of chromosomes 3, 5, 7, 9, 11, 15, 19, and 21. In both cases, these are associated with either direct or indirect dysregulation of a D-type cyclin, a unifying event in the pathogenesis of MM. Secondary genetic events include activating mutations of ras, translocations of myc, and inactivating mutations of p53. Recently, using an integrated genomic analysis of primary MM tumors and a panel of MM cell lines, we identified a promiscuous array of mutations that lead to activation of the NF-κB pathway. These include activating mutations (e.g., translocations, amplifications) of CD40, TACI, LTßR, NIK, NFKB1, and NFKB2, and inactivating mutations (e.g., biallelic deletions, point mutations) of TRAF2, TRAF3, cIAP1, cIAP2, and CYLD. All together, we estimate that about 20 percent of patients have mutations that activate the NF- κB pathway. The single most common mutation is TRAF3 in 13 percent of patients. As expected, TRAF3 inactivation is associated with stabilization of NIK and increased processing of NFKB2 p100 to p52. Introduction of wildtype TRAF3 into MM cell lines with TRAF3 inactivation results in decreased processing of p100 to p52, associated with decreased growth and viability. Using gene expression profiling, low levels of TRAF3 expression can be used as a surrogate marker to identify patients with inactivating mutation, who, in a randomized clinical trial, appear to be resistant to glucocorticoids, and, in contrast, markedly sensitive to proteasome inhibitors. This suggests that the NF-κB pathway is an important mediator of the activity of both these classes of drugs in MM and that developing clinically relevant assays to assess NF-κB activity is important. Although assessment of nuclear NFKB1 p50 and NFKB2 p52 can be done by immunohistochemistry, it is difficult to quantitate. We have found measuring the transcriptional effects of NF-κB activation by gene expression profiling to be a reliable assay. Using this, we find high levels of NF-κB activity in both normal plasma cells and MM samples with mutations, indicating that this is a critical pathway for normal PC biology that is co-opted by mutation in the malignant plasma cells of some patients.

Translational Research in Myeloid Disorders

Dennis D. Hickstein, MD, National Cancer Institute, National Institutes of Health, Bethesda, MD

Mary C. Dinauer, MD, PhD, Wells Center for Pediatric Research, Indiana University School of Medicine, Indianapolis, IN
Gene Therapy of Chronic Granulomatous Disease: What’s Next?

Chronic granulomatous disease is an inherited disorder of innate immunity in which phagocytic leukocytes are unable to generate microbicidal oxidants due to mutations in any one of four genes encoding subunits of the superoxide-generating NADPH oxidase. Life-threatening, recurrent bacterial and fungal infections, as well as inflammatory granulomas, are the hallmarks of the disease. Mutations in an X-linked gene encoding the gp91^phox subunit account for approximately two-thirds of cases. Murine models of CGD established by knockout technologies resemble the human disease, and are useful for assessment of efficacy and safety of hematopoietic stem cell (HSC) gene therapy. Pre-clinical studies in murine X-CGD established that retroviral-mediated gene transfer of gp91^phox ameliorates the defects in host defense and resolution of the inflammatory response. These studies also suggested that even partial correction of oxidase activity in a minority of phagocytes will be of at least some clinical benefit. The ectopic expression of gp91^phox outside of its normal expression in neutrophils and monocyte/macrophages had no obvious adverse consequences. Recent studies in murine X-CGD showed that nonmyeloablative irradiation is effective as conditioning for engraftment of transduced HSC, although this may bias which transduced marrow cells contribute to long-term hematopoiesis. NADPH oxidase activity can also be reconstituted by retroviral- or lentiviral-mediated gene transfer to human CGD marrow in vitro and in xenograft transplants. The first Phase 1 clinical trials in which recipients received CD34+ cells transduced with retroviral vectors demonstrated proof of concept in that peripheral blood neutrophils with reconstituted NADPH oxidase activity were detected, albeit at only very low levels (<1 percent) and disappearing within nine months post-infusion. This was not surprising given that patients did not receive any preparative regimen, and there is no intrinsic selection for gene-corrected cells in CGD. A recent Phase I clinical trial utilized a partially myeloablative dose of busulfan conditioning in combination with an improved ex vivo transduction protocol and a retroviral vector with an LTR with increased potency in myeloid cells. Higher initial levels of (12-31 percent) of oxidase-positive neutrophils were followed by an unexpected further increase in the number of gene-corrected neutrophils that, importantly, was not associated with an increase in total neutrophil numbers. Analysis of vector integration sites revealed expansion of myeloid clones harboring activating insertions in three growth-promoting genes, MDS1/EVI1, PRDM16, and SETBP1. A new trial for X-CGD being conducted at the NIH is also utilizing conditioning with a partially ablative busulfan regimen but a different retroviral vector backbone. Updates on ongoing clinical trials at the NIH and in Germany will be presented. Current pre-clinical studies are focused on development of enhancer-deleted vectors with improved safety profiles and their utilization in the context of nonmyeloablative conditioning protocols.

Dennis D. Hickstein, MD, National Cancer Institute, National Institutes of Health, Bethesda, MD
Development of New Therapeutic Approaches to Leukocyte Adhesion Deficiency

Children with the genetic immunodeficiency leukocyte adhesion deficiency (LAD) have heterogeneous molecular defects in the leukocyte integrin CD18 molecule and suffer life-threatening bacterial infections due to the inability of their leukocytes to adhere and migrate to sites of infection. Hematopoietic stem cell transplant remains the only curative therapy for LAD, however, the toxicity of this treatment has limited its use in genetic diseases such as LAD. We tested new stem cell transplant and gene therapy approaches to LAD in a canine model of LAD. Matched littermate transplant following a non-myeloablative conditioning regimen with 200 cGy total body irradiation resulted in reversal of the phenotype of CLAD with minimal toxicity. However, the optimal therapy for LAD would involve gene therapy since no donor is required, and graft-versus-host disease is not an issue. We first evaluated ex vivo gammaretroviral-mediated gene therapy using two non-myeloablative conditioning regimens – 200 cGy TBI or 10 mg/kg busulfan – with or without post-transplant immunosuppression. Six of 11 treated CLAD dogs achieved therapeutic levels of CD18+ leukocytes. Conditioning with either TBI or busulfan allowed long-term engraftment, and immune suppression was not required for efficacy. The percentage of CD18+ leukocytes increased over 6-8 months to levels ranging from 0.72 percent to 8.37 percent at one-year follow-up in the 6 dogs. These levels resulted in reversal of the severe CLAD phenotype. Linear amplification-mediated-PCR assays indicated polyclonality of insertion sites. Since gammaretroviral vectors have led to insertional activation of nearby oncogenes and leukemia in previous gene therapy trials, we carried out gene therapy in the CLAD model using a vector based on foamy virus (FV). Four of 5 CLAD dogs receiving non-myeloablative conditioning with 200 cGy TBI and infusion of autologous CD34+ hematopoietic stem cells transduced by the FV vector expressing canine CD18 had complete reversal of the CLAD phenotype, which was sustained two years following treatment. In vitro assays demonstrated correction of the lymphocyte proliferation and neutrophil adhesion defects that characterize CLAD. There were no genotoxic complications, and integration site analysis demonstrated polyclonal marking by transduced cells. These results suggest that FV vectors will be effective in treating human hematopoietic diseases such as LAD. These studies indicate that new therapeutic approaches are becoming available to treat LAD now, 20 years after the initial cloning of the CD18 cDNA.

George A. Diaz, MD, PhD, Mount Sinai School of Medicine, New York, NY
Chemokine Receptor Mutations and Neutrophil Trafficking Defects in WHIM Syndrome

Granulopoiesis in the bone marrow proceeds rapidly and continuously processes from immature myeloblasts through the release of mature polymorphonuclear neutrophils (PMNs). The pool of circulating plus marginal neutrophils is approximately equal to the pool of bone marrow myeloid precursors. In congenital neutropenias in which myeloid maturation is compromised, the pool of myeloid precursors in the bone marrow is relatively intact, but the pool of mature circulating and marginal PMNs is diminished. In recent years, genetic dissection of these disorders has allowed the elucidation of molecular mechanisms of myeloid development. In contrast, neutropenias resulting from trafficking defects (i.e., failure of PMNs to reach the circulating/marginal compartment) have not been well described. Using in vivo murine models, the contributions of specific chemokine receptors to myeloid cell trafficking have been explored and two receptors in particular, CXCR4 and CXCR2, have been suggested to play critical and opposing roles. One of these receptors, CXCR4, has been extensively characterized because of its role as a co-receptor used during infection by some strains of the human immunodeficiency virus. Study of a rare congenital neutropenia and combined immunodeficiency known as WHIM (Warts, Hypogammaglobulinemia, Immunodeficiency, and Myelokathexis) syndrome has revealed that the condition is caused by mutations in CXCR4 that impair receptor downregulation. While some studies of the disease pathophysiology suggested that accelerated apoptosis might underlie the neutropenia, characterization of mutant receptor function suggests a more important role for enhanced interactions with bone marrow stromal cells via increased signaling through the chemokine receptor following ligand binding. Thus, the characteristic accumulation of mature PMNs in the bone marrow seen in the myelokathexis of WHIM syndrome is an example of peripheral neutropenia caused by impaired PMN trafficking. Interestingly, genetic analysis of patients who have the full WHIM phenotype has revealed disease phenocopies lacking mutations in the CXCR4 gene. Ongoing study of these patients, as well as those with isolated myelokathexis, will be an important resource for the identification of additional genes that are critical in the regulation of neutrophil trafficking through the bone marrow, vascular, and marginal compartments.

Targeting Transcription Factors

Steven Grant, MD, Massey Cancer Center, Virginia Commonwealth University, Richmond, VA

Jay L. Hess, MD, PhD, University of Michigan, Ann Arbor, MI
Overview of Transcription Factor Targets in Leukemia

Acute Myelogenous Leukemia (AML) is the most common form of acute leukemia in adults. It is characterized by a block in myeloid differentiation. As transcription factors play critical roles in normal self-renewal, differentiation, and apoptosis, a prediction is that disruption of transcription factor pathways will play an important role in AML. The first set of transcription factor targets that were described were the products of chromosome translocations, examples being PML/RAR alpha and AML1/ETO. While there are therapies available targeting PML/RAR alpha, many questions remain relating to the pathogenesis of induction of leukemia, and in other cases no targeted therapy exists. A second set of transcription factor targets include those that play a role in normal myeloid differentiation, including C/EBP alpha and PU.1. These pathways are disrupted in AML by multiple mechanisms, and we have begun to understand how drugs can and do target these pathways. A third set of targets includes genes thought to play a role in myeloid development, but their precise role is not clear. This includes the Hox genes and RAR family members. Finally, a fourth class, which includes members of the polycomb and trithorax families, has been implicated in leukemia, including AML. Their roles in inducing epigenetic changes, in leukemogenesis, and approaches to targeting them in AML treatment represent exciting new areas in leukemia research.

John H. Bushweller, PhD, University of Virginia, Charlottesville, VA
Targeting RUNX1 in Leukemia

The subunits of the heterodimeric transcription factor CBF, core binding factor β (CBFβ) and RUNX1 (CBFα), play a critical role in hematopoiesis. Knockouts of either Cbfb or Runx1 are embryonic lethal with a profound block in hematopoietic development. The RUNX1 subunit binds to DNA and recruits coactivators or corepressors in a context-dependent manner. CBFβ binds to the Runt domain of RUNX1, increasing the DNA-binding of the RUNX1 subunit 20-40 fold and protecting RUNX1 against ubiqitination and subsequent proteasome degradation. Chromosomal rearrangements that target the core-binding factor genes are some of the most common mutations in leukemia. RUNX1 (AML1) is disrupted by the t(8;21)(q22;q22), t(12;21)(p13;q22), t(3;21)(q26;q22), t(16;21)(q24;q22), t(1;21)(p36;q22), t(5;21)(q13;q22), t(12;21)(q24;q22), t(14;21)(q22;q22), t(15;21)(q22;q22), and t(17;21)(q11.2;q22), all of which are associated with myeloid and lymphocytic leukemia. Based on the importance of the interaction between CBFβ and RUNX1 for normal CBF function, it is not surprising that this interaction also plays a critical role in the function of CBF translocations. The introduction of point mutations into the Runt domain in AML1-ETO, product of the t(8;21), which abrogate CBFβ binding 400-fold, results in loss of the ability to immortalize lin^- BM cells as well as a loss of leukemogenesis in a mouse model of AML1-ETO leukemia. This validates this protein-protein interaction as an appropriate target for therapeutic development. Using virtual screening, i.e., computational docking of compounds to the CBFβ binding interface on the structure of the RUNX1 Runt domain, we have identified weak affinity lead inhibitors for the protein-protein interaction between the RUNX1 Runt domain and CBFβ. Fluorescence resonance energy transfer (FRET) and ELISA assays have been used to characterize these compounds. Binding to the Runt domain has been confirmed by saturation transfer difference (STD) NMR spectroscopy. Subsequently, we have synthesized a library of compounds around these initial leads to sample the structure-activity relationships (SAR) and optimize these compounds. This has resulted in the development of several compounds with low micromolar IC₅₀ values. These compounds inhibit the growth of the t(8;21) cell lines Kasumi-1 and SKNO-1. With Kasumi-1 cells, we have also shown significantly increased apoptosis in the presence of inhibitor. Treatment of Kasumi-1 cells with inhibitor in combination with ATRA results in a synergistic increase in the differentiation of Kasumi-1 cells, supporting direct targeting of AML1-ETO as the mechanism of action rather than off-target effects. These compounds represent the first small molecule inhibitors targeting RUNX1 and inhibiting its interaction with CBFβ. Future efforts are directed at further optimization and testing of such compounds in appropriate mouse models.

Michael Thirman, MD, University of Chicago, Chicago, IL
Targeting MLL Fusion Genes in Leukemia

11q23 translocations result in rearrangement of the MLL gene and occur frequently in both ALL and AML. More than 50 different recurring cytogenetic aberrations that affect the MLL gene at 11q23 have been described. The critical feature of these chromosomal rearrangements is the generation of a chimeric protein consisting of the N-terminus of MLL fused to the C-terminus residues encoded by the gene on the partner chromosome. MLL partner proteins can be classified based on their subcellular localization. The cytoplasmic partners contribute an oligomerization domain that acts to dimerize the N-terminus of MLL. The critical transforming property of the nuclear partners of MLL remains elusive. Recent data suggest that the protein-protein interactions of the nuclear partner are critical to leukemogenesis. Previously, we showed that the EAF1 interaction domain of ELL is necessary and sufficient for leukemogenesis mediated by MLL-ELL. In addition, the interaction of AF10 with the histone methylase DOT1 is essential for leukemic transformation by MLL-AF10. Recently, the MLL partner proteins AF4, ENL, AF9, and AF10 have been identified in a complex that stimulates transcriptional elongation by recruitment of P-TEFb kinase and by mediating histone H3-K79 methylation via DOT1. In view of the poor outcomes observed with standard chemotherapy, efforts to develop new therapeutic strategies have focused on the targets that are critical for leukemogenesis as defined using model systems. For example, RNA interference has been directed at the sequence found at the point of fusion between MLL and AF4. In addition, inhibition of expression of HOXA9, a gene upregulated in MLL-associated leukemia, has also been examined. To inhibit activated FLT3, a frequent cooperating pathway in MLL-associated leukemia, small molecule inhibitors have also been examined. Peptide-based therapeutics are an attractive approach to deliver macromolecules across the cell membrane. Cationic protein transduction domains can be used to deliver peptides that target specific protein-protein interactions in a concentration-dependent, receptor-independent manner. Recently, a cell-penetrating peptide that blocks the interaction between AF4 and AF9 has been shown to inhibit the proliferation of MLL-AF4 but not MLL-AF9 cell lines. To develop targeted therapy directed against MLL fusion proteins, we have examined the interaction of MLL with menin, which is essential for the initiation and maintenance of MLL-associated leukemias in experimental models. We have developed a transducible peptide that contains the TAT domain fused to MLL sequences which block the interaction of MLL with menin. To determine the effect of this MLL peptide on the growth of leukemia cells, we have transduced MLL-AF9 and MLL-ELL leukemia cells with the TAT-MLL peptide and observed significant effects on viability. We have examined a series of N- and C-terminal truncated MLL peptides to define the minimal domain required to disrupt the MLL-menin interaction. Compared to control peptides, TAT-MLL inhibits proliferation of MLL leukemic cell lines with modest inhibitory effects on cell lines that do not express MLL fusion genes. Future advances will likely require targeting multiple components of the MLL-partner protein complex using both peptide and small molecule inhibitors.

Personalized Medicine in Hematologic Disorders

Ching-Hon Pui, MD, St. Jude Children’s Research Hospital, Memphis, TN

Brian Gage, MD, Washington University Medical School, St. Louis, MO
Pharmacogenetic-Based Warfarin Therapy

Clinicians can estimate the therapeutic warfarin dose by genotyping patients for single nucleotide polymorphisms (SNPs) that affect warfarin metabolism or sensitivity. SNPs in the cytochrome P450 complex (CYP2C9) affect warfarin metabolism: Patients who have the CYP2C9*2 and/or CYP2C9*3 variants metabolize S-warfarin slowly and are two-to-three times more likely to have an elevated international normalized ratio (INR) or hemorrhage during warfarin initiation than those without these variants. Each CYP2C9*2 allele is associated with a 19 percent reduction in the therapeutic warfarin dose, and each CYP2C9*3 allele is associated with a 33 percent reduction in the therapeutic warfarin dose. To prevent overdosing poor metabolizers, clinicians should initiate warfarin carefully in patients known to have these variants. Furthermore, dose adjustments after two, three, or four days of warfarin therapy should be more cautious in poor metabolizers to prevent a supratherapeutic INR the following week. SNPs in vitamin K epoxide reductase (VKORC1) correlate with warfarin sensitivity. Patients who are homozygous for a common VKORC1 promoter polymorphism, –1639 G>A (also designated as VKOR 3673, haplotype A, or haplotype*2), have lower levels of VKORC1 mRNA expression, resulting in greater warfarin sensitivity. Per A allele, these patients require 28 percent less warfarin, an association that is reflected in INR values obtained after three or four days of warfarin therapy. Algorithms that combine these SNPs with information available before warfarin is initiated (e.g., age, body size, use of amiodarone, and smoking status) explain half or more of the variability in warfarin dose. An algorithm that combines these pharmacogenetic factors with an INR after the third warfarin dose is even more accurate. [We have made these algorithms available on a nonprofit Web site, www.warfarindosing.org, which estimates the therapeutic warfarin dose.] Although non-randomized studies suggest that this approach can prevent supratherapeutic INR values during warfarin initiation, whether it will prevent adverse events is unknown. Also unknown is whether the proposed pharmacogenetics approach is cost effective.

Co-Authors: Charles Eby, MD, Elena Deych, MS, Petra Lenzini, MS, Paul Milligan, PharmD, Gloria Grice, PharmD, and Susan Gatchel, Washington University Medical School, St. Louis, MO

References:
Rieder MJ, Reiner AP, Gage BF, et al. Effect of VKORC1 haplotypes on transcriptional regulation and warfarin dose. N Engl J Med. 2005;352:2285-93.

Millican E, J-Lenzini PA, Milligan PE, et al. Genetic-based dosing in orthopaedic patients beginning warfarin therapy. Blood. 2007;110:1511-5.

Russell E. Ware, MD, PhD, St. Jude Children’s Hospital, Memphis, TN
Individualized Therapy in Sickle Cell Disease

The complex pathophysiology of sickle cell disease (SCD) involves anemia, hemolysis, and vaso-occlusion, leading to a broad spectrum of acute and chronic organ damage. An estimated 100,000 persons in the United States and millions worldwide have SCD, most of whom are homozygous for the HbS beta-globin mutation (HbSS, β^s/β^s). Despite having a similar or identical genotype, persons with SCD exhibit substantial phenotypic variability in their hematological parameters, clinical manifestations, and treatment responses. Over the past 10-15 years, hydroxyurea has emerged as an important therapeutic option for SCD. Although the mechanisms of action by which hydroxyurea is beneficial in SCD have not been fully elucidated, salutary treatment effects derive from increasing fetal hemoglobin (HbF) levels, lowering leukocyte count, and reducing hemolysis as measured by serum lactate dehydrogenase (LDH). In prospective trials, hydroxyurea treatment resulted in a wide variability in therapeutic response in children and adults. When hydroxyurea therapy is titrated to maximum tolerated dose (MTD), which varies between 15-35 mg/kg/day, many children are "high responders" with substantial increases in HbF and decreases in leukocyte count and serum LDH, while some are relative "low responders" despite apparent treatment compliance. Predictors of the HbF response to hydroxyurea at MTD include the baseline percentHbF, hemoglobin concentration, leukocyte count, and reticulocyte count, as well as the actual MTD value. Genetic modifiers of the HbF response to hydroxyurea include several polymorphisms in the ARG2, FLT1, AQP9, CYP2C9, MAP3K5, TOX, HAO2, PDE7B, NOS1 and NOS2a genes, plus the 6q22.2-23.2 and 8q11-q12 linkage peaks. Additional genetic polymorphisms could potentially affect hydroxyurea pharmacokinetics through effects on drug absorption, metabolism, and clearance; proposed candidate genes include urea transporters, plasma enzymes, the TGF-β/BMP family, and selected hepatic metabolic pathways. Many genes could potentially affect hydroxyurea pharmacodynamics such as ribonucleotide reductase, erythroid transcription factors, the TGF-β family, and genes affecting neutrophils and adhesion molecules, among others. The UGT1A1 promoter polymorphism does not directly influence the HbF response but does significantly affect the bilirubin concentration while on hydroxyurea therapy. Individualized therapy is not yet a reality for patients with SCD, but the field is moving forward with the advances in genetic analysis and the availability of stored DNA samples from prospective clinical trials. Future pharmacogenomic studies should provide important clues regarding treatment response to hydroxyurea and other therapeutic options for patients with SCD.

Stella M. Davies, MBBS, PhD, Cincinnati Children’s Hospital and Medical Center, Cincinnati, OH
Pharmacogenetics in Acute Leukemia

Important progress has been made in the treatment of children with leukemia in the last 30 years, such that most children with ALL and a majority of children with AML are now cured of their disease. Despite this major advance, failure of therapy, including treatment-related mortality, relapse of disease, and second malignancy still occurs in children receiving identical regimens to children who are cured. Understanding the variable response to drugs seems particularly pressing in the field of leukemia, in which the stakes are high (failure to cure usually leads to death), drugs commonly have a narrow therapeutic index, and toxicities can be severe (a significant frequency of toxic death is a feature of most AML protocols, for example). In one of the first examples of pharmacogenetics in oncology, Weinshelboum and colleagues identified polymorphic responses to the key anti-leukemic drug, 6-mercaptopurine (6-MP), in 1980, over 25 years ago, and polymorphism of the gene thiopurine S-methyl transferase (TPMT) remains one of the best understood examples of pharmacogenetic variation. TPMT is a cytosolic drug-metabolizing enzyme that catalyzes the S-methylation of 6-MP and azathioprine. In their original seminal study, Weinshilboum and Sladek demonstrated a very clear tri-modal frequency of TPMT activity in red blood cells from 298 unrelated control adults. One in 300 subjects lacks TPMT activity, and 11 percent have intermediate levels. Family studies showed that the frequency distribution was due to inheritance. While phenotypic studies have shown a clear tri-modal distribution, the genetic basis of phenotypic variation has proved more complex. Seventeen variant TPMT alleles have been identified to date, although three variant alleles account for the majority (>95 percent) of persons with intermediate (one variant allele) or low (two variant alleles) TPMT activity. Subsequent clinical studies have demonstrated very clearly that TPMT polymorphism can predict toxicity of 6-MP and effectiveness of therapy. Children with ALL with intermediate or absent TPMT activity are at higher risk of myelosuppression when prescribed standard doses of 6-MP. In addition, patients with low TPMT activity are at increased risk of secondary cancers. Despite elegant studies demonstrating the role of TPMT phenotype in response to 6-MP and the easy availability of predictive genotyping, incorporation into clinical practice has been slow, and proof of benefit of therapy adjustment in a randomized controlled trial has proved difficult. The complexity of predictive genotyping for a leukemia treatment schedule that includes multiple drugs metabolized by multiple genes is apparent, and, as high throughput genotyping is now easily feasible, it is evident that the challenge in developing a predictive genotype is more statistical than technical. In addition, pharmacogenetic studies of childhood AML show that predictive genotypes may be dependent on the drugs used or on the dose employed, increasing the complexity of personalizing therapy for leukemia. Future directions in the clinical application of personalized therapy for leukemia will be facilitated by genome wide approaches to gene discovery, sophisticated statistical approaches to gene-gene interactions, and concerted efforts to develop approaches applicable outside of the setting of specialized research centers.

Platelet Granule Biogenesis and Secretion

Leslie V. Parise, PhD, University of North Carolina, Chapel Hill, NC

Sidney W. Whiteheart, PhD, University of Kentucky College of Medicine, Lexington, KY
Platelet Secretion: The Role of the SNARE Proteins and Their Regulators

Upon activation, platelets release hundreds of different proteins and small molecules, many of which are important for hemostasis, but several may play roles in the sequelae of thrombus formation. Platelets contain three types of secretory compartments or granules: dense core, alpha, and lysosomes. Secretion of cargo from each compartment requires integral membrane proteins called SNAREs (Soluble NSF Attachment Protein Receptors). v-SNAREs from the granules and heterodimeric t-SNAREs from the plasma membrane (or open canalicular system) form a trimeric complex that spans the two membranes to mediate fusion and cargo release. Using a permeabilized-platelet secretion assay and specific antibodies, inhibitory peptides, and recombinant proteins, it has been possible to assign specific t-SNAREs to each granule secretion event. The syntaxin 4/SNAP-23 heterodimer mediates release from alpha granules and lysosomes; syntaxin 2/SNAP-23 plays a role in all three release events. Knockout mice have been used to assign roles for the v-SNAREs. Platelets contain four v-SNAREs (VAMP-2, -3, -7, and -8), but only VAMP-8/endobrevin is required. Deletion of VAMP-8 causes a significant defect in cargo release from all three granules; however, in its absence, either VAMP-2 or -3 can inefficiently compensate. Consistent with this ex vivo analysis, in vivo studies of laser-induced vascular injury show that VAMP-8 null animals have a clear defect in both the extent and rate of thrombus formation. Because of their defects, the VAMP-8 null animals will be useful in probing the role of platelet exocytosis in processes potentially associated with thrombosis (i.e., atherosclerosis and angiogenesis). These studies complete the characterization of the platelet’s core exocytic apparatus but open the door to future work on the regulation of SNAREs and how the platelet activation cascades interface with the secretory machinery. A number of syntaxin regulators have been identified in platelets but their roles are only now being uncovered. Munc18 proteins (isoforms a, b, and c) serve as syntaxin chaperones and are essential for platelet secretion. Munc13 proteins (isoforms 1 and 4), which "prime" t-SNAREs for v-SNARE binding, are also important for platelet granule release. Munc13 proteins are of specific interest due to their calcium and diacylglcerol binding sites. Tomosyn, recently discovered in platelets, may affect v-/t-SNARE interactions. Future studies need to focus on characterizing other regulatory elements of the platelet secretory machinery. Uncovering the molecular events required for platelet granule release will undoubtedly lead to the development of better anti-thrombotic drugs.

Co-Authors: Robert Flaumenhaft, MD, PhD, and Gwenda G. Graham, PhD, Beth Israel Deaconess Medical Center, Boston, MA; and Qiansheng Ren, University of Kentucky, Lexington, KY

Joseph Italiano, PhD, Brigham and Women’s Hospital, Boston, MA
Angiogenesis is Regulated by a Novel Mechanism: Pro- and Anti-Angiogenetic Proteins are Organized into Separate Platelet α-Granules and Differentially Released

In addition to their "classic" role in hemostasis, platelets are now known to be major contributors in wound healing and tumor growth. Stored within the α-granules is an array of angiogenic regulatory proteins, which are deposited by the secretion reaction of surface-activated platelets into the local environment of a tumor or wound. However, because platelet α-granules contain both stimulators and inhibitors of angiogenesis, how secretion could be used to selectively stimulate or inhibit angiogenesis remains unclear. We have found that resting platelets contain distinct populations of α-granules that segregate by pro- or anti-angiogenic properties. Immunofluorescence and immunoelectron microscopy reveal that pro- and anti-angiogenic proteins are in separate and distinct α-granules of platelets. VEGF (an angiogenesis stimulator) and endostatin (an angiogenesis inhibitor) localize to separate and distinct α-granules. Similarly, double immunofluorescence microscopy for thrombospondin-1, basic FGF, fibrinogen, and von Willebrand factor confirmed that these proteins segregate into distinct and separate α-granules. These observations motivate the hypothesis that distinct α-granules might undergo differential release during platelet activation. Treatment of human platelets with a selective PAR4 agonist (AYPGKF-NH₂) in vitro stimulated the release of endostatin and suppressed the release of VEGF-containing granules. In contrast, a selective PAR1 agonist (TFLLR-NH₂) stimulated the release of VEGF in vitro, but not endostatin. Thus, platelets contain their angiogenesis regulatory proteins in pharmacologically and morphologically distinct populations of α-granules, which are regulated by differential release upon platelet activation. The role of generating and sorting these different α-granules, thus, falls to the megakaryocyte, and immunofluorescence microscopy reveals the presence of distinct populations of α-granules that are sorted and delivered to platelets and proplatelets. Future studies will characterize the molecular mechanisms regulating differential packaging of pro- and anti-angiogenic regulatory proteins in megakaryocytes and the identification of the signaling pathways that regulate differential granule release.

Co-Authors: Judah Folkman, MD, and Giannoula Klement, MD, Children’s Hospital Boston and Harvard Medical School, Boston, MA

William A. Gahl, MD, PhD, National Human Genome Research Institute, National Institutes of Health, Bethesda, MD
Platelet Organelles: Windows to Function and Disease

Specific human diseases prompted us to study platelet organelles, including alpha granules, dense bodies, and Golgi. Gray Platelet syndrome (GPS) patients lack alpha granules, and we sought to learn where within platelets alpha granule proteins reside in the absence of an alpha granule compartment. We first determined the contingent of proteins in the alpha granules of normal platelets. We prepared subcellular fractions from human platelets using sucrose gradient ultracentrifugation. Fraction 2 contained GP1b and represented cell surface and OCS membrane proteins. Fraction 6 contained alpha granules, as verified by Glut-3 labeling on immuno-electron microscopy (IEM). After polyacrylamide gel electrophoresis, LC MS/MS identified 219 non-redundant, non-mitochondrial proteins in fraction 6. These included 65 percent of the 81 proteins previously identified by others in the platelet releasate, and 44 new alpha granule proteins. Of particular interest, IEM confirmed the presence of Scamp2, APLP2, ESAM, and LAMA5 in platelet alpha granules for the first time. We are now examining GPS platelet fractions using similar proteomic analyses. Are alpha granule proteins uniformly distributed throughout the GPS platelet, are they degraded, or are they re-distributed to the plasma membrane or another membrane? These data will put into context the function of the GPS gene, whose identity is being pursued as well. Studies of dense granules grew out of our interest in Hermansky-Pudlak syndrome (HPS), a disorder of oculocutaneous albinism and delta storage pool deficiency. Eight different genes can cause different subtypes of HPS, all of which lack dense bodies but have normal alpha granules, meaning that at some point the genesis of alpha and delta granules diverges. Many investigators pursued how the HPS gene products form delta granules out of extant membranes. The HPS-2 gene encodes the beta-3A subunit of adaptor complex-3. Other HPS proteins interact with each other in biogenesis of lysosome-related organelles complexes or BLOCs; HPS1 and HPS4 form BLOC-3; HPS3, HPS5, and HPS6 comprise BLOC-2; HPS7 and HPS8 are components of BLOC-1. The findings were obtained through studies of lysosomes in fibroblasts and melanosomes in melanocytes, but they apply to delta granules in megakaryocytes. White Platelet Syndrome is characterized by the retention of the Golgi in mature platelets. Its genetic defect is being pursued. All of these disorders can be investigated using molecular tags to follow intracellular organelle formation and movement within megakaryocytes cultured from normal and affected patients. Proteomic studies can also be performed in these cells.

Co-Authors: Meral Gunay-Aygun, MD, Amanda Helip-Wooley, PhD, and Dawn M. Maynard, PhD, National
Human Genome Research Institute, National Institutes of Health, Bethesda, MD; Harry Heijnen, PhD, University
Medical Center Utrecht, Netherlands; and James G. White, MD, University of Minnesota, Minneapolis, MN

Leukemia Stem Cells

Craig T. Jordan, PhD, University of Rochester Medical Center, Rochester, NY

Scott Armstrong, MD, PhD, Children’s Hospital Boston, Boston, MA
Origins of Leukemia Stem Cells

Cellular heterogeneity is a characteristic of human acute myelogenous leukemias (AML) and also of myeloid leukemias that arise in genetically defined murine models. Detailed assessment of populations of cells with unique immunophentotypes has demonstrated that an isolatable subset of cells is capable of initiating leukemia in recipient mice. These leukemia-initiating cells possess high proliferative potential and are capable of giving rise both to cells that can further propagate the disease and to more differentiated cells incapable of propagating leukemia. As these properties are reminiscent of normal hematopoietic stem cells (HSC), this cellular population is frequently referred to as leukemia stem cells (LSC). Human AML stem cells frequently express CD34 and lack expression of CD38 (CD34+/CD38-). As human HSC are CD34+/CD38-, this suggests that LSC are related to the normal HSC counterpart. However, antigens not normally expressed on human HSC, such as CD123 (Interleukin-3 receptor), can be identified on LSC. Furthermore, it is likely that the immunophenotype of LSC varies between patients and potentially between subtypes of AML. Such heterogeneity prompts the question whether all AMLs arise from HSC or if other more differentiated cells can be the cells of origin of AML. Furthermore, if there is heterogeneity in the cells of origin for AML, might there be classes of oncogenes that initiate leukemias from specific cell types? These questions are currently being assessed in murine models of AML. Retroviral transduction of either HSC or more committed progenitor cells with translocation-associated fusion oncogenes leads to the development of AML supporting the possibility that multiple cell types are capable of being the cell of origin in AML. Certain fusion oncoproteins, such as BCR-ABL, are only capable of initiating disease from HSC, whereas fusion oncoproteins, such as MLL-fusions or MOZ-TIF2, can initiate AML from either HSC or committed progenitors. Detailed characterization of these murine leukemias demonstrates that LSC can possess an immunophenotype characteristic of more differentiated cells, while simultaneously expressing a gene expression program associated with HSC. Thus, it appears that the LSC are "partial" stem cells that express an HSC program in the context of more differentiated hematopoietic cells. These experiments demonstrate that LSC can be generated from committed progenitors without widespread reprogramming of gene expression, and a stem-cell-associated program is activated in the process. Furthermore, they highlight the fact that some LSC may be quite different from normal HSC providing hope that LSC can be specifically targeted. Ongoing studies will determine if therapeutic targeting of such cells is possible and will define the critical genes and gene expression programs necessary for LSC proliferation and survival.

Catriona Jamieson, MD, PhD, University of California, San Diego Medical Center, Moores Cancer Center, San Diego, CA
Dissecting the Molecular Evolution of Chronic Myeloid Leukemia Stem Cells

A growing body of evidence suggests that a primitive population of self-renewing cancer stem cells (CSC) is responsible for therapeutic resistance and relapse. As a result of landmark discoveries that have helped to unravel its molecular pathogenesis, chronic myeloid leukemia (CML) represents an important paradigm for understanding the molecular evolution of CSC, particularly in the context of leukemia (leukemia stem cells; LSC). CML is a myeloproliferative disorder that without treatment or sometimes despite it progresses inexorably from a relatively well-tolerated chronic phase to a therapeutically recalcitrant blast crisis phase. In a study involving 100 CML blood and marrow samples, we found that progression to blast crisis was typified by expansion of the granulocyte-macrophage progenitor (GMP) pool. Blast crisis phase CML GMP had increased BCR-ABL transcripts endowing them with a proliferative advantage as well as activation of β-catenin, a self-renewal gene, resulting in enhanced in vitro replating capacity. Moreover, transplantation of immunocompromised (RAG2-/-γ_c-/-) neonatal mice with blast crisis CML GMP resulted in robust leukemic engraftment in primary and secondary recipients, suggesting that the GMP population was enriched for LSC. To address the controversy regarding the nature and hematopoietic developmental stage of β-catenin activating mutations in CML, genomic DNA and cDNA Wnt mediator sequencing analysis was performed on normal (n=49) or CML (n=51) hematopoietic stem cells (HSC), GMP and lineage-positive or blast populations. Although there were no identifiable activating mutations in b-catenin, APC, axin 1, LEF-1, cyclin D1, or c-myc, there was recurrent missplicing of GSK3β (GSK3βdel) in blast crisis GMP that was not evident in normal progenitors or CML blasts. Moreover, GSK3β protein expression by myeloid progenitors decreased with progression from chronic to blast crisis phase CML. Lentiviral luciferase transduced GSK3βdel blast crisis myeloid progenitors gave rise to BCR-ABL-positive, activated β-catenin expressing progenitors in vivo as well as granulocytic sarcomas and robust secondary bioluminescent engraftment. Finally, lentivirally enforced expression of GSK3βdel in luciferase-expressing chronic phase progenitors enhanced their bioluminescent engraftment, suggesting that missplicing of negative regulators of the Wnt/β-catenin pathway may be a novel mechanism for enhancing LSC self-renewal that may be more broadly applicable to CSC in other malignancies.

Allison Blair, PhD, BSc, Bristol Institute for Transfusion Sciences, Bristol, United Kingdom
Acute Lymphoblastic Leukemia Stem Cells

Childhood acute lymphoblastic leukemia (ALL), the most common paediatric cancer, is a heterogeneous disease, composed of different genetic, clinically relevant subtypes. The different ALL subtypes have been considered to represent clonal expansion of malignant cells that had been transformed at different maturation stages within the haemopoietic hierarchy. Therefore, the degree of maturation of the initial transformed target cell would influence the characteristics of resulting leukemia blasts. Optimization of therapy for childhood ALL has resulted in vastly improved survival rates of around 80 percent. However, a significant proportion of patients relapse, often with disease that is highly refractory to further therapeutic intervention. Only a minority of ALL cells are thought to have the ability to act as stem cells in vivo to maintain the leukemic clone. This sub-population may exhibit decrease