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Antimicrobial peptides (AMPs) play an important role in the host defense against various microbes. One of the most efficient human AMPs is the human beta defensin-3 (hBD-3) which is produced by, e.g. keratinocytes and lung epithelial cells. However, the structure-function relationship for AMPs and in particular for defensins with their typical three disulfide bonds is still poorly understood. In this study the importance of the three disulfide bonds for the activity of the AMPs is investigated with biological assays and with biophysical experiments utilizing different membrane reconstitution systems. The activities of natural hBD-3, hBD-3-c (cyclic variant with one disulfide bond), and hBD-3-l (linear variant without disulfide bonds) and fragments thereof were tested against specific Gram-negative bacteria. Furthermore, hemolytic and cytotoxic activities were analyzed as well as the potency to neutralize immune cell stimulation of lipopolysaccharide (LPS). Experiments using reconstituted lipid matrices composed of phospholipids or LPS purified from the respective Gram-negative bacteria, showed that the membrane activity of all three hBD-3 peptides is decisive for their capability to kill bacteria and to neutralize LPS. In most of the test systems the linear hBD-3-l showed the highest activity. It was also the only peptide significantly active against polymyxin B-resistant Proteus mirabilis R45. However, the stability of hBD-3 against protease activity decreases with decreasing number of disulfide bonds. This study demonstrates that the refining of AMP structures can generate more active compounds against certain strains.

Central nervous system infiltration and relapse are poorly understood in childhood acute lymphoblastic leukemia. We examined the role of zeta-chain-associated protein kinase 70 in preclinical models of central nervous system leukemia and performed correlative studies in patients. Zeta-chain-associated protein kinase 70 expression in acute lymphoblastic leukemia cells was modulated using short hairpin ribonucleic acid-mediated knockdown or ectopic expression. We show that zeta-chain-associated protein kinase 70 regulates CCR7/CXCR4 via activation of extracellular signal-regulated kinases. High expression of zeta-chain-associated protein kinase 70 in acute lymphoblastic leukemia cells resulted in a higher proportion of central nervous system leukemia in xenografts as compared to zeta-chain-associated protein kinase 70 low expressing counterparts. High zeta-chain-associated protein kinase 70 also enhanced the migration potential towards CCL19/CXCL12 gradients in vitro. CCR7 blockade almost abrogated homing of acute lymphoblastic leukemia cells to the central nervous system in xenografts. In 130 B-cell precursor acute lymphoblastic leukemia and 117 T-cell acute lymphoblastic leukemia patients, zeta-chain-associated protein kinase 70 and CCR7/CXCR4 expression levels were significantly correlated. Zeta-chain-associated protein kinase 70 expression correlated with central nervous system disease in B-cell precursor acute lymphoblastic leukemia, and CCR7/CXCR4 correlated with central nervous system involvement in T-cell acute lymphoblastic leukemia patients. In multivariate analysis, zeta-chain-associated protein kinase 70 expression levels in the upper third and fourth quartiles were associated with central nervous system involvement in B-cell precursor acute lymphoblastic leukemia (odds ratio=7.48, 95% confidence interval, 2.06–27.17; odds ratio=6.86, 95% confidence interval, 1.86–25.26, respectively). CCR7 expression in the upper fourth quartile correlated with central nervous system positivity in T-cell acute lymphoblastic leukemia (odds ratio=11.00, 95% confidence interval, 2.00–60.62). We propose zeta-chain-associated protein kinase 70, CCR7 and CXCR4 as markers of central nervous system infiltration in acute lymphoblastic leukemia warranting prospective investigation.

Signaling via the epidermal growth factor receptor (EGFR) pathway has emerged as one of the key mechanisms in the development of the central nervous system in Drosophila melanogaster. By contrast, little is known about the functions of EGFR signaling in the differentiated larval brain. Here, promoter-reporter lines of EGFR and its most prominent activating ligands, Spitz, Keren, and Vein, were used to identify the brain structures relevant for the EGFR pathway. Unexpectedly, promoter activity of all these pathway components was found in the mushroom bodies, which are known to be a higher brain center required for olfactory learning. We investigated the role of the EGFR pathway in this process by using different mutant larvae with reduced pan-neuronal EGFR signaling and those with reduced EGFR signaling in mushroom bodies only. Expression of a dominant-negative form of EGFR as well as silencing of the ligands via RNA interference was applied and resulted in significantly impaired olfactory learning performances. General defects in the ability to taste or smell as well as impaired EGFR signaling during embryonic development could be excluded as major reasons for this learning phenotype. In addition, targeted expression of a constitutively active form of the ligand Spitz also led to a significantly reduced learning ability. Thus, very low levels as well as very high levels of EGFR signaling are deleterious for olfactory learning and memory formation. We hypothesize that EGFR signaling in a certain range maintains a homeostatic situation in the mushroom bodies that is necessary for proper learning and memory.

Tunicates comprising a wide variety of different species synthesize antimicrobial peptides as important effector molecules of the innate immune system. Recently, two putative gene families coding for antimicrobial peptides were identified in the expressed sequence tag database of the tunicate Ciona intestinalis. Two synthetic peptides representing the cationic core region of one member of each of the families displayed potent antibacterial and antifungal activities. Moreover, the natural peptides were demonstrated to be synthesized and stored in distinct hemocyte types. Here, we investigated the presence of these natural peptides, namely Ci-MAM-A and Ci-PAP-A, in the tunic of C. intestinalis considering that the ascidian tunic is a body surface barrier exposed to constant microbial assault. Furthermore, as the tunic may represent a major route of entry for pathogen invasion after its damage we monitored the location of these peptides upon a local inflammatory-like reaction induced by injection of foreign cells. Using immunocytochemistry and electron microscopy both peptides were localized to the tunic and were massively present in granulocytes of inflamed tissue. Conclusively, antimicrobial peptides may constitute a chemical barrier within the tunic of urochordates.

Patients with t(1;19)-positive acute lymphoblastic leukemia (ALL) are prone to central nervous system (CNS) relapses, and expression of the TAM (Tyro3, Axl, and Mer) receptor Mer is upregulated in these leukemias. We examined the functional role of Mer in the CNS in preclinical models and performed correlative studies in 64 t(1;19)-positive and 93 control pediatric ALL patients. ALL cells were analyzed in coculture with human glioma cells and normal rat astrocytes: CNS coculture caused quiescence and protection from methotrexate toxicity in Mer(high) ALL cell lines, which was antagonized by short hairpin RNA-mediated knockdown of Mer. Mer expression was upregulated, prosurvival Akt and mitogen-activated protein kinase signaling were activated, and secretion of the Mer ligand Galectin-3 was stimulated. Mer(high) t(1;19) primary cells caused CNS involvement to a larger extent in murine xenografts than in their Mer(low) counterparts. Leukemic cells from Mer(high) xenografts showed enhanced survival in coculture. Treatment of Mer(high) patient cells with the Mer-specific inhibitor UNC-569 in vivo delayed leukemia onset, reduced CNS infiltration, and prolonged survival of mice. Finally, a correlation between high Mer expression and CNS positivity upon initial diagnosis was observed in t(1;19) patients. Our data provide evidence that Mer is associated with survival in the CNS in t(1;19)-positive ALL, suggesting a role as a diagnostic marker and therapeutic target.

Ci-MAM-A24, a synthetic antimicrobial peptide derived from a peptide precursor from immune cells of the marine invertebrate Ciona intestinalis, has been shown to be potently active against representatives of Gram-positive and Gram-negative bacteria by permeabilising their cytoplasmic membrane. In the present study, the activity of Ci-MAM-A24 against different bacterial pathogens frequently causing therapeutic problems was tested. In particular, the killing capacity of Ci-MAM-A24 against clinically important anaerobic bacteria as well as multiresistant aerobic strains such as meticillin-resistant Staphylococcus aureus (MRSA), vancomycin-resistant enterococci, extended-spectrum beta-lactamase-producers and multiple-resistant Pseudomonas aeruginosa strains was monitored. Virtually all strains proved to be highly susceptible to Ci-MAM-A24 at low concentrations [minimum bactericidal concentration (MBC)

Arenicin-1 (Ar-1) is a β-sheeted antimicrobial peptide from the marine lugworm Arenicola marina. To elucidate the significance of its unique 18-residue cyclic structure and of six cationic arginines for its biological activity and its interaction with biomembranes, we synthesized one linear peptide in which the two cysteines were exchanged for serines (C/S-Ar-1) and a cyclic peptide in which all arginines were replaced by lysines (R/K-Ar-1). We addressed antibacterial and hemolytic activities, the impact of the peptides on bacterial morphology, and their binding to, intercalation into, and permeabilization of model membranes composed of phospholipids or lipopolysaccharide (LPS). In accordance with high salt concentration in sea water, the antibacterial activity of Ar-1 was almost insensitive to high NaCl concentrations. In contrast, the linear derivative lost activity under these conditions against polymyxin B-resistant Proteus mirabilis. Ar-1 intercalated into phospholipid and LPS membranes and formed heterogeneous and short-lived lesions. However, when the peptide was present in both membrane leaflets, it formed defined pores. This characteristic was not observed for the linear derivative C/S-Ar-1. Apparently, the disulfide bond provides conforma-tional stability, which has an impact on salt tolerance, prevents fast degradation by trypsin, and is a prerequisite for the formation of structurally defined pores.

A novel gene family coding for putative antimicrobial peptides was identified in the EST (expressed sequence tag) database of the sea squirt Ciona intestinalis, and one of these genes was molecularly cloned from the Northern European Ciona subspecies. In situ hybridization and immunocytochemical analysis revealed that the natural peptide is synthesized and stored in a distinct haemocyte type, the univacuolar non-refractile granulocytes. By semiquantitative RT–PCR (reverse transcription–PCR) analysis, it was shown that the expression of the gene is markedly up-regulated in haemocytes after immune challenge. To evaluate the antimicrobial potency of the putative defence protein, we synthesized a peptide corresponding to its cationic core region. The peptide was highly effective against Gram-negative and Gram-positive bacteria including several human and marine pathogens as well as the yeast Candida albicans. Notably, the antibacterial activity of the peptide was retained at salt concentrations of up to 450 mM NaCl. Using two different methods we demonstrated that the peptide kills Gram-negative and Gram-positive bacteria by permeabilizing their cytoplasmic membranes. CD spectroscopy revealed that, in the presence of liposomes composed of negatively charged phospholipids, the peptide undergoes a conformational change and adopts an α-helical structure. Moreover, the peptide was virtually non-cytolytic for mammalian erythrocytes. Hence, the designed salt-tolerant antimicrobial peptide may represent a valuable template for the development of novel antibiotics.

Constitutive activation of the FMS-related tyrosine kinase-3 (FLT3) is a common feature of acute leukemias which can be caused by activating mutations or by high expression levels. Aberrant FLT3 downstream signaling is mediated via the RAS/MAPK, PI-3-Kinase/AKT, and STAT5 pathways leading to proliferation, survival, and therapy resistance. In MLL-rearranged acute lymphoblastic leukemia (ALL) activating FLT3 tyrosine kinase domain mutations (TKDs) affecting codons D835 and I836 are frequently identified (Armstrong et al., 2003; Taketani et al., 2004). In addition, MLL-rearranged ALLs are consistently characterized by an exceptionally high FLT3 expression (Armstrong et al., 2002) which has been shown to be associated with ligand independent signaling (Stam et al., 2005). However, the prognostic impact of a constitutive activation of FLT3 in this ALL subset remains controversial. Here, we report on the FLT3 mutational status and gene transcription levels in a large cohort of 95 infants and 72 children with MLL-rearranged ALL. Results obtained were further complemented by a high resolution melting (HRM) screen for common activating NRAS and KRAS mutations at codons 12, 13, and 61. All patients were enrolled in the multicenter trials ALL-BFM 86, 90, 95, 2000, and AIEOP-BFM ALL 2009 as well as Interfant-99 and Interfant-06. In infants, FLT3-TKD mutations were identified in 12/95 patients (12.6%) including one novel insertion/deletion involving codons D835 to S838. In only 2/95 patients (2.1%) an alteration in the juxtamembrane domain of FLT3 was detected. Of the 12 infants with mutation only 2 suffered from a relapse, 2-years cumulative incidence of relapse (CIR) 18%± 12%. In children, only one FLT3 aberration (FLT3-TKD D835 mutation) was detected (1/72, 1.4%). FLT3 transcript levels were analyzed by quantitative real-time PCR in 124 patients (69 infants and 55 children) with available RNA. When we separated the infant cohort into two groups according to the median RQ value, FLT3high and FLT3low, the CIR was significantly different (CIR 19%±7% vs. 66%± 9%, Gray p=0.0001). Of the 6 patients with low FLT3 transcription level, but with presence of a mutation, only one had a relapse. These results indicate that activating FLT3 mutations may compensate for the high relapse risk of patients with a low FLT3 expression. Accordingly, we could show that the CIR was significantly reduced for infants with a low FLT3activation (low transcription, no mutation 19%±7%) compared to those with a high FLT3 activation (high transcription or mutation 75%±9%, Gray p The only other known recurrent mutations in pediatric MLL-rearranged ALL are activating N- and KRAS mutations at codons 12, 13, and 61 (Liang et al., 2006; Andersson et al., 2015). As RAS signaling is considered as a putative downstream target of FLT3, we investigated the frequency of these mutations and their impact in the context of the prognostic value of FLT3 activation. We identified non-synonymous N/KRAS mutations in 21/95 (22.1%) infants and in 10/72 (13.9%) children. The presence of activating RAS mutations correlated with a higher rate of relapse and a lower probability of event-free survival (pEFS). For infants alone, constitutive activation of N/KRAS resulted in a lower pEFS (43%±6% wt vs. 11%±8%, p=0.01), but there were no significant differences in the CIR (40%±6% wt vs. 51±12%, p=0.40). In summary, we confirm that MLL-rearranged infant ALL represents a biologically distinctive entity with unique molecular genetic features. However, in contrast to published studies, we report that hyperactivation of FLT3 signaling is associated with a good prognosis in MLL-rearranged infant ALL. Our data has important implications for the design of rational therapies in MLL-rearranged ALL as the use of small molecule FLT3 inhibitors may be disadvantageous in some infants depending on FLT3 expression levels and FLT3 and RAS mutational status. Figure 1. Cumulative incidence of relapse (CIR) at 3 years for infant MLL-rearranged ALL patients with high or low FLT3 activation. Figure 1. Cumulative incidence of relapse (CIR) at 3 years for infant MLL-rearranged ALL patients with high or low FLT3 activation. Disclosures No relevant conflicts of interest to declare.

Most children with B-cell precursor ALL (BCP-ALL) have an excellent prognosis but some patients are susceptible to CNS relapse, the mechanisms of which are largely unknown. Zap-70 is a tyrosine kinase mainly expressed in normal T- and NK-cells, but it is also expressed at low levels in normal B-cells. Zap-70 is however overexpressed in B-cell chronic lymphocytic leukemia (B-CLL) and is associated with an aggressive course of the disease. In B-CLL, Zap-70 was shown to up-regulate the expression of CCR7 (Calpe et al., 2011), which in turn allows T-lymphoblasts to enter the CNS (Buonamici et al., 2009). We hypothesized that Zap-70 mediated upregulation of CCR7 may enhance the homing and the survival of CNS-prone BCP-ALL cells in the CNS niche. We first transduced the 697 BCP-ALL cell line with a lentiviral vector carrying a non-targeting GFP (shGFP) or a Zap-70 (shZap-70) specific shRNA. Down-regulation of Zap-70 resulted in a significantly reduced CCR7 mRNA and surface protein expression. 697shGFP and 697shZap-70 cells were then injected intravenously into NSG mice, which were sacrificed when leukemic symptoms developed. There were no statistically significant differences in leukemic bone marrow infiltration and survival between the groups. However, immunohistochemical scoring of CNS infiltration (Krause et al., 2015) revealed that 9/10 animals (90%) in the 697shGFP group were CNS+ and only 2/7 animals (29%) in the 697shZap-70 group showed a CNS+ status (p = 0.0345). This suggests that Zap-70 mediated reduction in CCR7 impairs the CNS-positive phenotype of 697 cells in vivo. We next investigated if the Zap-70/CCR7 axis influences the ability of primary samples from patients to infiltrate the CNS of xenografts. Zap-70 mRNA was measured in pediatric BCP-ALL patients and patients with Zap-70 expression levels higher than the median were considered Zap-70high, and the remaining patients Zap-70low. 1 x 106 cells of 5 Zap-70high and 5 Zap-70low samples were injected intrafemorally into duplicate NSG mice. Interestingly, 7/10 (70%) of the mice injected with Zap-70high cells showed a CNS+ phenotype, whereas only 1/10 (10%) of the mice bearing Zap-70low samples were CNS+ (p = 0.0198). Importantly, leukemic cells of one Zap-70high patient purified from the xenograft CNS showed a marked upregulation of CCR7 expression as compared to cells isolated from bone marrow or spleen. These data suggest that the Zap-70/CCR7 axis is beneficial for patient BCP-ALL cells in the CNS of NSG mice. To test whether Zap-70/CCR7 is associated with CNS involvement in patients, we analyzed Zap-70 and CCR7 mRNA expression in diagnostic primary material of 76 BCP-ALL patients. The cohort contained 21 CNS-positive and 55 CNS-negative patients and was previously published by Cario et al. (2007). Patients were grouped into either Zap-70high/Zap-70low or CCR7high/CCR7low groups according to expression levels higher or lower than the median of the respective gene. Zap-70 expression was highly correlated with CCR7 expression (p = 0.0003; Spearman r = 0.401). There were no statistically significant differences in outcome surrogates such as prednisone response or MRD and there was no correlation between Zap-70/CCR7 expression and outcome (relapse or death). However, a trend towards high CCR7 levels and the occurrence of death could be observed (p = 0.055). Interestingly, high expression of Zap-70 or CCR7 correlated with the presence of blasts in the cytospin of the initial cerebrospinal fluid sample (p = 0.014 and p = 0.045, respectively). Furthermore, there was a significant correlation between high Zap-70 and a higher CNS status and a trend between high CCR7 and a higher CNS status according to AIEOP-BFM 2009 criteria (p = 0.024 and p = 0.098, respectively). We finally performed comparative correlations in 48/76 patients (63.2%) that were Zap-70high/CCR7high (n=24) or Zap-70low/CCR7low (n=24). Most importantly, a significant correlation between Zap-70high/CCR7high expression and a higher CNS status could be observed (p = 0.009). These data suggest that a high expression of Zap-70 or CCR7 is associated with CNS infiltration in BCP-ALL patients and that measurements of Zap-70 and CCR7 expression should be combined. Altogether, we show that Zap-70/CCR7 is important for BCP-ALL cells to enter the CNS and to survive in that niche. We propose Zap-70/CCR7 as a mechanism of CNS leukemia and a diagnostic marker with potential therapeutic implications. Disclosures No relevant conflicts of interest to declare.

In vertebrate development the Dickkopf protein family carries out multiple functions and is represented by at least four different genes with distinct biological activities. In invertebrates such as Drosophila and Caenorhabditis, Dickkopf genes have so far not been identified. Here we describe the identification and characterization of a Dickkopf gene with a deduced amino acid sequence closely related to that of chicken Dkk-3 in the basal metazoan Hydra. HyDkk-3 appears to be the only Dickkopf gene in Hydra. The gene is expressed in the gastric region in nematocytes at a late differentiation stage. In silico searches of EST and genome databases indicated the absence of Dkk genes from the protostomes Drosophila and Caenorhabditis, whereas within the deuterostomes, a Dkk-3 gene could be identified in the genome of the urochordate Ciona intestinalis. The results indicate that at an early stage of evolution of multicellularity Dickkopf proteins have already played important roles as developmental signals. They also suggest that vertebrate Dkk-1, 2 and 4 may have originated from a common ancestor gene of Dkk-3.

MLL-fusion is the most common genetic abnormality in acute lymphoblastic leukemia (ALL) of infancy and occurs in approximately 80% of the cases. Infant ALL represents a biologically distinctive entity with a highly immature pro-B immunophenotype associated with a particularly unfavorable prognosis due to a high proportion of early relapses. This may be due to the survival of dormant residual disease protected by the bone marrow niche. We have characterized a pair of monozygotic twin sisters diagnosed with ALL in early infancy. Tumor cells from both children carried the t(11;19) translocation (MLL-ENL fusion) and both patients were treated according to the ALL-BFM 2000 protocol. Despite good initial treatment responses in both twins, one sister developed very early relapse (twin A) and succumbed to the disease. The other went into continuous complete remission (twin B) and, as of today, is alive after 9 years. The clinical data of the 2 patients are presented in Table 1. Diagnostic bone marrow (BM) aspirates of both sisters were injected into the femoral bones of NOD SCID gamma (NSG) mice. Survival of xenografted mice bearing twin A was significantly longer due to a lower proliferative capacity of twin A as compared to twin B cells. In vivo Bromodeoxyuridine (BrdU) assays revealed that twin B cells were proliferating equally fast in BM and spleen, while in twin A cells, the BM markedly suppressed entry into S-phase. Flow cytometry from xenograft BM identified a large CD34+ population in twin A cells that was almost absent in twin B cells. Furthermore, BM of twin A xenografts showed a CD34+/CD38-/CD19- stem cell like population undetectable in twin B animals. Most interestingly, injection of minimal residual disease (MRD) negative remission bone marrow (PCR for Ig-gene rearrangements and the MLL-fusion gene) of both sisters into NSG mice resulted in a full-blown xenograft leukemia in animals bearing twin A but not twin B. Taken together, these data suggest that fatal relapse in twin A may be due to a quiescent stem cell like population kept in check by unknown mechanisms. Next, we performed gene expression analyses on xenografted leukemias from twin A (initial, leukemia amplified from remission BM, relapse) and from twin B (initial). STRING analysis of gene expression differences revealed that, amongst other findings related to cell cycle regulation and DNA-repair, twin A cells downregulated genes indicating a reduced interferon pathway activity (CXCL10, IFI30, TRAIL, STAT1, OAS1, MX1) and several genes connected to TYRO protein kinase binding protein (TYROBP) shown previously to regulate the activity of natural killer (NK) cells. This suggests that twin A cells may evade immunosurveillance as a mechanism of disease persistence. 51Chromium-release assays with IL-2 stimulated allogeneic NK cells from two healthy donors and the xenografted twin cells showed that twin A cells were significantly less sensitive to NK cell mediated lysis as compared to twin B cells. Whether the tumor cells also display differential susceptibility to antibody-dependent cell-mediated cytotoxicity is currently investigated. While the extensive genomic characterization of the twin leukemias is under way, we show evidence from xenograft experiments, gene expression profiles and functional in vitro experiments that dormant residual cells in MLL-rearranged ALL can evade immunosurveillance. These findings serve as a rationale to employ immunotherapeutic approaches in infant ALL patients in order to improve their dismal prognosis. Finally, we report the first monozygotic twin pair with MLL-rearranged ALL and discordant clinical outcomes. This provides a unique opportunity and a model to gain insight into the clonal composition of infant leukemia and the origins of leukemia relapse. | Parameter | Twin A | Twin B | | --------------------------- | -------------- | -------------- | | Age at diagnosis (days) | 98 | 147 | | WBC (initial)/µl | 334.000 | 103.000 | | Blasts (Peripheral Blood) % | 97 | 91 | | Blasts (BM) % | 98 | 97 | | CNS involvement | Not determined | Negative | | Immunophenotype | Pro-B | Pro-B | | Cytogenetics | t(11;19) | t(11;19) | | Prednisone-Response | Poor | Good | | Blasts (BM), day 15 % | 32 | 1 | | MRD day 33 | Negative | Negative | | MRD day 78 | Negative | 9 years | | Time to relapse | 227 days | n/a | | Overall survival | 390 days | >9 years | Table 1: Patient characteristics of the MLL-ENL positive twin pair. WBC, white blood cells; BM, bone marrow; CNS, central nervous system; MRD, minimal residual disease. *low positive, not quantifiable Disclosures No relevant conflicts of interest to declare.