ALBERT

Description: Chronic lymphocytic leukemia (CLL) is a monoclonal expansion of antigen-experienced human B cells. A key role for the B-cell antigen receptor (BCR) is suggested by the striking structural similarity among BCRs of unrelated patients. The goal of this study was to identify specific antigens or classes of antigens reacting with BCRs to better understand the role that such interactions play in the development, progression, and evolution of the disease. Since we and others recently described that CLL mAbs react with surfaces of apoptotic cells, we searched for reactivity of CLL mAbs with specific autoantigens associated with or created during apoptosis and other catabolic chemical processes. Reactivities of 28 recombinantly expressed CLL mAbs with defined autoantigens were studied using solid phase protein arrays. These experiments indicated significant reactivity with several intracellular autoantigens involved in systemic autoimmunity, some of which are displayed on the cell surface during apoptosis (e.g., Sm, Ku, snRNP A, BB¢, and C, and others). In addition, array analyses revealed reactivity with LDL modified by oxidation (oxidized-LDL) or derivatized with malondialdehyde (MDA-LDL). These data were provocative because, during apoptosis, cells can display oxidation-induced neoepitopes on lipoproteins, lipids, and proteins. In addition, mice immunized with syngeneic apoptotic cells can produce autoantibodies to oxidation-induced neoepitopes that result from conjugation to membrane molecules of metabolites of lipid peroxidation, such as MDA, 1-palmitoyl-2-(5-oxovaleroyl)-sn-glycero-3-phosphorylcholine (POVPC), and 4-hydroxynonenal (HNE). Therefore, we tested if CLL mAbs react with MDA-, POVPC-, and HNE-modified BSA. Next, we determined the extent of binding of the same mAbs with products of other metabolic processes linked to apoptosis, i.e., nitrosylation (nitrotyrosine-BSA) and glycosylation (advanced glycosylation end products, AGE-BSA). Seventeen of 28 mAbs reacted with nitrotyrosine and 9 with AGEs. All mAbs binding AGEs also interacted with MDA and all but one (16/17) nitrotyrosine-reactive mAbs bound MDA. Of note, the AGE-reactive mAbs exhibited greater reactivity with methylglyoxal- than carboxymethyllysine-modified BSA, two chemically defined AGEs. The subset of CLL mAbs reacting with apoptotic cells and neo-determinants generated by oxidation, nitrosylation, and glycosylation most frequently exhibits IGHV of minimal or no somatic mutations, However, even though the majority of CLL mAbs reactive with these epitopes were polyreactive and not somatically mutated, some unmutated mAbs failed to bind the targets and a few mutated mAbs did react. Thus, lack of somatic mutation does not necessarily confer autoantigen binding. In summary, our data suggest that CLL BCRs react with autoantigens associated with cellular apoptosis and chemical modifications of proteins and lipids that occur naturally in health and disease. The normal pre-immune repertoire is enriched in Abs reactive with the types of epitopes mentioned above. These “natural” antibodies serve housekeeping functions such as clearance of cellular debris and products of normal catabolism. They also represent the humoral arm of the innate-like B-cell immune responses, providing a first-line of defense against blood-borne pathogens. Therefore, our findings may have implications for understanding the normal cellular precursors of CLL cells.

Description: Diamond Blackfan anemia (DBA) is a rare inherited bone marrow failure syndrome characterized by red blood cell hypoplasia, congenital anomalies and cancer predisposition. In addition, short stature and poor skeletal growth are found in a subset of DBA patients, suggesting similar developmental abnormalities in erythropoiesis and osteogenesis in that subset. Furthermore it has been shown recently that osteoblasts secrete erythropoietin, linking the marrow niche to the modulation of erythropoiesis. DBA has been shown in the majority of cases to result from haploinsufficiency of large or small ribosomal subunit proteins. The p53 pathway, known to be activated by abortive ribosome assembly, contributes to the erythroid failure of DBA. We studied two DBA genotypes in vitro using murine embryonic stem (ES) cell lines harboring gene trap mutations in ribosomal proteins RPS19 and RPL5, respectively. Both mutants had decreased embryoid body (EB) formation, decreased definitive erythroid colony formation and similar p53-dependent primitive erythroid differentiation defects (see Figure A). Cell cycle analyses were normal in the Rps19 mutant ES cells, but there was a significant G2/M arrest in the Rpl5 mutant ES cells, which was unaffected by p53 knockdown. In addition, the Rpl5 mutant cells had a more pronounced growth defect in culture compared to the Rps19 mutant cells (Figure B). ES cells were differentiated, in vitro, to osteoblasts using established culture conditions, and confirmed both by morphology and molecular characterization (e.g. RUNX2 and Osteopontin). Following 14 days of osteogenic differentiation, bone mineralization was confirmed via Alizarin Red staining. A marked reduction in Alizarin Red staining was seen in the Rpl5 mutant cells while there was only a slight diminution of staining in the Rps19 mutant ES cultures (see Figure C). Therefore the erythroid differentiation defect appears similar in both the Rps19 and Rpl5 mutant ES cells. However the Rpl5 mutant appears to have a more severe phenotype at the ES stage, as evidenced by a pronounced p53-independent G2/M arrest and slower growth rate and subsequently during osteogenic differentiation. These data suggest an explanation for the more severe non-erythroid phenotype seen in a subset of DBA patients. Disclosures: No relevant conflicts of interest to declare.

Description: Severe sepsis is a leading cause of death and disability. Anemia in sepsis survivors affects close to 100% of patients after the third day of in-hospital stay, regardless of blood levels on admission. Circulating levels of Erythropoietin (Epo) are low; paradoxically, administration of recombinant Epo is ineffective, and related to increased morbidity. During sepsis, bone Marrow is hypoproliferative. While transfusions can improve outcome in the short term, its use increases the risk of infection and mortality without any sustained beneficial effect. The pathogenesis of anemia during sepsis is unclear. High mobility group box 1 (HMGB1), a cytokine that is a critical mediator of sepsis, is released into circulation a few days after sepsis onset, remaining increased for 8 weeks after severe sepsis. HMGB1 levels are increased for at least 8 weeks in murine models of sepsis survival. To induce severe sepsis, cecal ligation and puncture (CLP) was performed in BALB/c mice. Three days after CLP, mice developed persistent anemia, represented by a significant reduction in hematocrit (Sham=49.8±3.2 vs. CLP=29.7±6.7%; p≤0.001), hemoglobin (16.7±1.2 vs. 9.9±2.4mg/dL; p≤0.001), and red blood cells mass (10.2±0.7 vs. 5.4±1.7 x106/µL; p≤0.001). Anemia persisted for at least 25 days after CLP. In CLP survivors, reticulocyte counts were erratic, and insufficient to the degree and duration of anemia (8.2±0.8 vs. 6.6±2.1%; p=ns). Analysis of terminal erythroid differentiation using CD44 and Ter119 or CD44 and FSC as markers demonstrated a significant decrease in all erythroid progenitors, from proerythroblast to orthochromatic erythroblast. Concomitantly, mice surviving CLP developed splenomegaly. Splenic architecture was disrupted after CLP, with expansion of the red pulp, characteristic of stress erythropoiesis. Analysis of terminal erythroid differentiation demonstrated an increase in the quantity of erythroid progenitors. An anti-HMGB1 mAb (2G7) was administered after CLP. Strikingly, 2G7-treated septic mice were significantly protected from developing anemia, and had levels of hemoglobin and hematocrit similar to sham-operated mice. These results highlight a critical role for HMGB1 as key modulator of stress erythropoiesis in a murine model of sepsis survivors. To get further insight into the function of HMGB1 and translate our findings to the pathophysiology of human erythropoiesis, we used CD34+ cells derived from cord blood. Cord blood-derived CD34+ cells were incubated in MethoCult in the presence or not of HMGB1. HMGB1 induced a dose dependent decrease in CFU-E. In murine sepsis, there is a stepwise elevation of different redox forms of HMGB1, with an early increase in all-thiol (inflammatory), followed by a partially oxidized before a fully oxidized (with no known inflammatory activity) appears. At day 7, all-thiol HMGB1 reduced significantly the number of CFU-E, while the fully oxidized had no significant effect. At day 14, the number of BFU-E was reduced in the presence of HMGB1, and further decreased with all-thiol HMGB1. In conclusion, our findings suggest that CLP is a reproducible model to study anemia of sepsis. In mice surviving sepsis, stress erythropoiesis is consistently found. Administration of anti-HMGB1 monoclonal antibody reverses anemia of murine sepsis, demonstrating that HMGB1 can be a potential target in the anemia of sepsis survivors. Translating the findings to the human system, we found that HMGB1 impairs differentiation of CD34+ cells towards the BFU-E and CFU-E stages in colony formation assays, implying that HMGB1 might play a role early during differentiation. The redox status of HMGB1 is critical for its biological function, since its effects are not retrieved when HMGB1 is fully oxidized. Disclosures: No relevant conflicts of interest to declare.

Description: Fetal hemoglobin (HbF) is a known modifier of sickle cell disease (SCD) severity. KLF-1 is a regulator of the globin switch. It does so by increasing beta-globin production and up-regulating BCL11A, a repressor of HbF synthesis. Pomalidomide, a second generation immunomodulatory drug (IMiD), regulates HbF and F-cell production during erythropoiesis in human CD34+ cells. The mechanism by which pomalidomide enhances F-cell production is not well understood. In this study, CD34+ cells were obtained after purification of peripheral blood and positive selection and cultured using a three-phase in vitro liquid culture system which recapitulates erythropoiesis, including terminal differentiation and enucleation, in the presence of no drug, pomalidomide, hydroxyurea, or dimethyl sulfoxide (DMSO; vehicle control). Erythroid differentiation was assessed morphologically and by flow cytometry using the transferrin receptor and glycophorin A as markers of erythroid maturation. Flow cytometry was used to quantify F-cells. RT-qPCR was used to quantify mRNA expression of BCL11A, KLF-1, and gamma-globin. Western blot was used to measure the total expression levels of BCL11A. In this culture system pomalidomide increased F-cells more than hydroxyurea in both SCD and normal control erythroid cultures. There was a significant decrease in BCL11A expression levels, a repressor of HbF synthesis, with pomalidomide but not with hydroxyurea. This decrease was seen in both SCD and normal samples. KLF-1 was not affected by pomalidomide. These findings suggest a very different mechanism of action for pomalidomide versus hydroxyurea in increasing F-cell production. Pomalidomide appears to target the erythroid specific BCL11A but not the more pleiotropic transcription regulator KLF-1. Since the F-cell production was augmented in the presence of pomalidomide in controls as well as SCD erythroid cultures this study suggests a role for pomalidomide in the pharmacologic augmentation of fetal hemoglobin levels, perhaps in addition to hydroxyurea, not only in SCD but in any beta-hemoglobinopathy. Disclosures: Chan: BioTheryX Inc: Employment.

Description: Key Points Pomalidomide selectively targets BCL11A and SOX6 to induce γ-globin synthesis. The mechanism of action of pomalidomide during erythropoiesis is independent of IKZF1 degradation, in contrast to multiple myeloma.

Description: Long-term memory formation depends on the expression of immediate early genes (IEGs). Their expression, which is induced by synaptic activation, is mainly regulated by the 3′,5′-cyclic AMP (cAMP)-dependent protein kinase/cAMP response element binding protein (cAMP-dependent protein kinase (PKA)/ cAMP response element binding (CREB)) signaling pathway. Synaptic activation being highly energy demanding, neurons must maintain their energetic homeostasis in order to successfully induce long-term memory formation. In this context, we previously demonstrated that the expression of IEGs required the activation of AMP-activated protein kinase (AMPK) to sustain the energetic requirements linked to synaptic transmission. Here, we sought to determine the molecular mechanisms by which AMPK regulates the expression of IEGs. To this end, we assessed the involvement of AMPK in the regulation of pathways involved in the expression of IEGs upon synaptic activation in differentiated primary neurons. Our data demonstrated that AMPK regulated IEGs transcription via the PKA/CREB pathway, which relied on the activity of the soluble adenylyl cyclase. Our data highlight the interplay between AMPK and PKA/CREB signaling pathways that allows synaptic activation to be transduced into the expression of IEGs, thus exemplifying how learning and memory mechanisms are under metabolic control.

Description: Abstract 799 Persistent research has begun to provide an understanding of the pathogenesis of B-cell chronic lymphocytic leukemia (CLL), a clonal expansion of a single B lymphocyte bearing a B-cell antigen receptor (BCR) with a monoclonal antibody (mAb) of defined amino acid sequence. Analyses of large collections of CLL mAb sequences have shown that many CLL clones express mAbs with very similar or stereotyped sequences. Indeed, almost 30% of CLL patients can be grouped on the basis of stereotyped sequences. This remarkable similarity is improbable by chance alone and therefore suggests that CLL BCRs are reacting to a common antigen(s), which may provide the stimulation necessary for the leukemic clone to survive and expand. We have been studying a large subset (at least 53 worldwide) of CLL mAbs that contain a rearranged heavy (H) chain encoded by IGHV1-69, IGHD3-16, and IGHJ3 with a stereotyped sequence (subset 6) and have shown that mAbs from this subset recognize non-muscle myosin heavy chain IIA (MYHIIA). In viable cells, MYHIIA resides in the cytoplasm as part of molecular motors involved in cell morphogenesis and locomotion. However, during apoptosis, MYHIIA structurally rearranges and becomes exposed on the cell surface, allowing interaction with CLL subset 6 mAbs. As assessed by immunohistochemistry of apoptotic cells generated spontaneously in a human T cell line (Jurkat), MYHIIA becomes exposed in large structures that do not contain condensed nuclear DNA. Using flow cytometry and staining with 7-amino-actinomycin and Annexin V-Phycoerytherin to determine early and late stages of apoptosis, we found that MYHIIA exposure (and concomitant CLL subset 6 mAb reactivity) occurred in a subgroup of both early and late apoptotic cells. Because some CLL mAbs can bind apoptotic cells, we wondered if these mAbs bound autoantigens revealed in the subgroup of apoptotic cells that exposed MYHIIA. The revealed autoantigen(s) could be MYHIIA, other proteins such as vimentin or filamin B, or chemical modifications such as oxidized epitopes, which are induced and exposed during apoptosis. Using flow cytometry, we measured the binding of MYHIIA exposed apoptotic cells (MEACs) to a panel of CLL mAbs that included those in stereotyped subsets (18/26) with only two from subset 6. The majority of CLL mAbs (16/26) bound MEACs well. Among these high binders, the majority (14/16) belonged to characterized stereotyped CLL mAb subsets. Interestingly, the level of MEAC binding inversely correlated with the degree of IGHV mutation in a statistically significant association (p 〈 0.0032). In CLL, IGHV mutation status associates with patient outcome, where patients with mutated (〉2%) IGHV tend to have longer survival. Because MEAC binding correlated with IGHV mutation, we investigated the association of MEAC binding with patient outcome. Of the 26 CLL mAbs, survival data were available for 24 patients. Remarkably, CLL mAbs with high binding to MEACs (n = 15) correlated with shorter patient survival (median survival = 99 months), whereas CLL mAbs with low binding to MEACs (n = 9) correlated with longer patient survival (median survival not reached). This association was statistically significant (p 〈 0.0087). In comparison, the association with patient survival and IGHV mutation status (18 unmutated and 6 mutated) did not reach statistical significance in this patient cohort (p 〈 0.0582). Thus, many stereotyped and non-stereotyped CLL mAbs recognize autoantigenic targets on MEACs, the subset of apoptotic cells exposing MYHIIA. Therefore CLL cells with these (or other) binding characteristics could be expanded in vivo, ultimately leading to poor patient outcome. Consistent with this hypothesis, our initial findings suggest that autoantigen (e.g., MEAC) binding, frequently found in members of stereotyped subsets, provides prognostic information for CLL patients regardless of IGHV mutation status and may better indicate patient outcome than IGHV mutation status. This possibility is provocative and requires further investigation with more mAbs from more patients. However if born out, this would be consistent with the notion that BCR binding and signaling can alter CLL cell biology and may represent the fact that IGHV mutation status implies the ability of the CLL BCR to bind antigen, whereas reactivity with MEACs is a true indicator of CLL BCR binding to antigen(s). Disclosures: No relevant conflicts of interest to declare.

Description: B-cell chronic lymphocytic leukemia (B-CLL) is a disease caused by the clonal expansion of a B lymphocyte expressing a unique monoclonal antibody (mAb) of a defined amino acid sequence. At least 27% of B-CLL patients share mAbs with very similar sequences, suggesting common antigen reactivity. A frequently occurring subset of B-CLL patients (at least 49 worldwide) carries an unmutated mAb with a nearly identical or stereotypic sequence that is characterized by a heavy (H) chain encoded by IGHV1-69, IGHD3-16, and IGHJ3 and a light (L) chain generally encoded by IGKV3-20. mAbs from this stereotypic subset (called subset 6) bind a characteristic cytoplasmic protein in human cells. We have recently shown that this protein is non-muscle myosin heavy chain IIA (MYHIIA). Because MYHIIA is an intracellular protein, we hypothesized that MYHIIA becomes exposed on the cell surface during apoptosis, allowing the mAb of the B-CLL cell to interact with MYHIIA. In this report, we provide evidence that subset 6 mAbs recognize MYHIIA-containing apoptotic cell structures. Apoptosis of the human T cell line (Jurkat), either spontaneous, or induced by treatment with camptothecin was assessed phenotypically by nuclear DNA rearrangement revealed by immunohistochemical staining with propidium iodide (PI). Costaining with rabbit anti-human MYHIIA followed by fluorescein isothiocyanate (FITC) labeled donkey anti-rabbit IgG revealed that MYHIIA becomes exposed during apoptosis in structures distinct from those containing fragmented nuclear DNA. PI costaining with recombinant subset 6 B-CLL IgG1 mAb and FITC labeled goat anti-human-IgG antibody shows that this B-CLL mAb binds similar apoptotic structures. Finally, Jurkat cells stained with subset 6 B-CLL mAb as before and rabbit anti-human MYHIIA followed by Rhodamine Red labeled donkey anti-rabbit IgG demonstrate that the B-CLL mAb colocalizes with MYHIIA containing structures. To further study this observation, Jurkat cell apoptosis was assessed by flow cytometry after staining with 7-AAD and Annexin V-Phycoerytherin (AV-PE). Costaining with anti-MYHIIA shows that anti-MYHIIA is exposed in both early (7-AAD+ AV-PE−) and late (7-AAD+ AV-PE+) apoptotic cells, but not in live (7-AAD−, AV-PE−) cells. Similarly, costaining with subset 6 B-CLL mAb demonstrates binding in both early and late apoptotic cells, but not live cells. Intriguingly, not all apoptotic Jurkat cells are MYHIIA+ or B-CLL mAb+. In summary, subset 6 B-CLL mAbs react with early and late apoptotic cells exposing MYHIIA. Reactivity with a specific apoptotic cell structure(s) may result in the maintenance and expansion of the leukemic clone.